ES2340459B1 - METHOD FOR DIAGNOSING OR DETERMINING THE GENETIC PREDISPOSITION TO DEVELOP HYPERTROPHIC MIOCARDIOPATIA. - Google Patents
METHOD FOR DIAGNOSING OR DETERMINING THE GENETIC PREDISPOSITION TO DEVELOP HYPERTROPHIC MIOCARDIOPATIA. Download PDFInfo
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Abstract
Método para diagnosticar o determinar la predisposición genética a desarrollar miocardiopatía hipertrófica.Method to diagnose or determine the genetic predisposition to develop cardiomyopathy hypertrophic
El método para diagnosticar o determinar la predisposición genética de un sujeto a desarrollar miocardiopatía hipertrófica (MCH), tanto familiar como esporádica, se basa en la identificación de mutaciones puntuales en el gen MYH7, que codifica la cadena pesada de la beta miosina cardíaca humana, asociadas con el desarrollo de MCH. Dicho método constituye una herramienta de diagnóstico útil que es particularmente importante cuando se estudian sujetos asintomáticos con sospecha de tener la enfermedad. Los sujetos asintomáticos procedentes de familias con una historia de MCH pueden ser estudiados selectivamente usando este método lo que permite disponer de un diagnósitco antes de la aparición de la enfermedad. A los individuos que posean la mutación asociada con la enfermedad se les pueden aconsejar pautas de comportamiento apropiadas.The method to diagnose or determine the genetic predisposition of a subject to develop cardiomyopathy hypertrophic (MCH), both familiar and sporadic, is based on the identification of point mutations in the MYH7 gene, which encodes the heavy chain of human cardiac beta myosin, associated with the development of MCH. Said method constitutes a tool of useful diagnosis that is particularly important when they study asymptomatic subjects with suspicion of having the disease. Asymptomatic subjects from families with a history of MCH can be studied selectively using this method as which allows to have a diagnosis before the onset of disease. To individuals who possess the mutation associated with the disease can be advised behavior patterns appropriate.
Description
Método para diagnosticar o determinar la predisposición genética a desarrollar miocardiopatía hipertrófica.Method to diagnose or determine the genetic predisposition to develop cardiomyopathy hypertrophic
La invención se relaciona con un método para determinar in vitro la predisposición genética de un sujeto a desarrollar miocardiopatía hipertrófica o para diagnosticar un sujeto de dicha enfermedad, basado en un ensayo genético, extracorpóreo, de una muestra biológica procedente de dicho sujeto, que comprende detectar la presencia o ausencia de una mutación puntual de un nucleótido, asociada con dicha enfermedad, en el gen MYH7 que codifica la cadena pesada de la beta miosina cardíaca humana.The invention relates to a method for determining in vitro the genetic predisposition of a subject to develop hypertrophic cardiomyopathy or to diagnose a subject of said disease, based on an extracorporeal genetic test of a biological sample from said subject, which comprises detecting the presence or absence of a point mutation of a nucleotide, associated with said disease, in the MYH7 gene that encodes the human cardiac beta myosin heavy chain.
La miocardiopatía hipertrófica (MCH) es una enfermedad autosómica dominante que representa un grupo heterogéneo de enfermedades del músculo cardíaco cuyos síntomas y manifestaciones clínicas incluyen grados variables de hipertrofia y alteración de la función sistólica y diastólica. La MCH puede ser familiar (MCHF) o esporádica (MCHE). Los individuos afectados por dicha enfermedad pueden ser sintomáticos o asintomáticos. La prevalencia es de aproximadamente 1/500 en la población general y la consecuencia más seria es la muerte súbita de los individuos portadores y aparentemente sanos.Hypertrophic cardiomyopathy (MCH) is a autosomal dominant disease representing a heterogeneous group of heart muscle diseases whose symptoms and clinical manifestations include varying degrees of hypertrophy and impaired systolic and diastolic function. The MCH can be family (MCHF) or sporadic (MCHE). The individuals affected by Such disease can be symptomatic or asymptomatic. The prevalence is approximately 1/500 in the general population and the most serious consequence is the sudden death of individuals bearers and seemingly healthy.
La MCHF es una enfermedad hereditaria del músculo cardíaco que se caracteriza por un incremento en la masa ventricular y una alteración de la función sistólica y diastólica. La patología de la MCHF es el resultado de las consecuencias fisiológicas de la alteración de la función sistólica y diastólica, y sus características patológicas están bien establecidas (Marón BJ & Epstein SE. 1980. Amer. J. Cardiol. 45:141-154; Braunwald E. 1997. Heart Disease: a textbook of cardiovascular medicine. WB Saunders; Philadelphia. p 1404). Además del hallazgo clásico del engrasamiento asimétrico del septo interventricular también puede observarse la hipertrofia de la pared anterior del ventrículo izquierdo y del apex cardíaco. La distribución anatómica y la severidad de la hipertrofia pueden ser muy variables. Con frecuencia se observa fibrosis en el ventrículo hipertrofiado y en la región del septo.MCHF is a hereditary disease of the heart muscle characterized by an increase in mass ventricular and an alteration of systolic and diastolic function. The pathology of the MCHF is the result of the consequences physiological disorders of systolic and diastolic function, and its pathological characteristics are well established (Marón BJ & Epstein SE. 1980. Amer. J. Cardiol. 45: 141-154; Braunwald E. 1997. Heart Disease: a textbook of cardiovascular medicine. WB Saunders; Philadelphia p 1404). In addition to the classic finding of asymmetric greasing of interventricular septum hypertrophy of the anterior wall of the left ventricle and cardiac apex. The Anatomical distribution and severity of hypertrophy can be very variable Fibrosis is often seen in the ventricle hypertrophied and in the region of the septum.
La mayoría de las anormalidades histológicas características de la MCHF tienen que ver con la desorganización de las miofibrillas en los miocitos. Los miocitos pueden hipertrofiarse hasta diez o veinte veces el diámetro normal de una célula cardíaca (Becker AE. 1989. Pathology of Cardiomyopathies, en Cardiomyopathies: Clinical Presentation, Differential Diagnosis, and Management, Shaver JA ed., F. A. Davis Co., New York, pp. 9-31; Braunwald E. 1997. Heart Disease: a textbook of cardiovascular medicine.WB Saunders; Philadelphia. p 1404).Most histological abnormalities characteristics of the MCHF have to do with the disorganization of Myofibrils in myocytes. Myocytes can hypertrophy up to ten or twenty times the normal diameter of a heart cell (Becker AE. 1989. Pathology of Cardiomyopathies, in Cardiomyopathies: Clinical Presentation, Differential Diagnosis, and Management, Shaver JA ed., F. A. Davis Co., New York, pp. 9-31; Braunwald E. 1997. Heart Disease: a textbook of cardiovascular medicine.WB Saunders; Philadelphia p 1404).
Actualmente se admite que la MCH es el resultado de mutaciones en genes que codifican las proteínas del aparato contráctil. De hecho, entre las posibles causas de dicha enfermedad, se reconoce la existencia de mutaciones en los siguientes genes: cadena pesada de la beta miosina cardíaca humana (MYH7); Proteína de unión a la miosina (MYBPC3); Troponina T (TNNT2); Troponina I (TNNI3); Troponina C (TNNC1); Alfa tropomiosina (TPM1), Cadena reguladora ligera de la miosina (MYL2); Cadena ligera esencial de la miosina (MYL3), Actina cardíaca alfa (ACTC) (Richard P et al; 2003 Circulation. 107(17):2227-32); Titina (TTN) (Satoh et al. 1999. Biochem Biophys Res Commun 262, 411); Cadena pesada de la alfa miosina (MYH6) (Tanigawa et al. 1990.) Cell 62,991); Sub-unidad no catalítica gamma 2, activada por AMP, de la proteína kinasa (PRKAG2) (Gollob et al. (2001) N Engl J Med 344, 1823; Blair et al. 2001 Hum Mol Genet 10, 1215); Quinasa 2 de la cadena ligera de la miosina (MYLK2) (Davis et al. 2001 Cell 107, 631); Genoma mitocondrial (Arbustini et al. Heart 1998; 80:548-58).It is currently recognized that MCH is the result of mutations in genes that encode the proteins of the contractile apparatus. In fact, among the possible causes of this disease, the existence of mutations in the following genes is recognized: human cardiac beta myosin heavy chain (MYH7); Myosin binding protein (MYBPC3); Troponin T (TNNT2); Troponin I (TNNI3); Troponin C (TNNC1); Alpha tropomyosin (TPM1), Light myosin regulatory chain (MYL2); Essential myosin light chain (MYL3), Cardiac actin alpha (ACTC) (Richard P et al ; 2003 Circulation. 107 (17): 2227-32); Titina (TTN) (Satoh et al . 1999. Biochem Biophys Res Commun 262, 411); Heavy chain of alpha myosin (MYH6) (Tanigawa et al . 1990.) Cell 62,991); Non-catalytic sub-unit gamma 2, activated by AMP, of the protein kinase (PRKAG2) (Gollob et al . (2001) N Engl J Med 344, 1823; Blair et al . 2001 Hum Mol Genet 10, 1215); Myosin light chain kinase 2 (MYLK2) (Davis et al . 2001 Cell 107, 631); Mitochondrial genome (Arbustini et al . Heart 1998; 80: 548-58).
Diversos análisis genéticos han permitido identificar mutaciones en el gen MYH7, que codifica la cadena pesada de la beta miosina cardíaca humana, asociadas a la aparición de MCHF (Seidman CE & Seidman JG. 1991. Mol. Biol. Med. 8:159-166; Tanigawa et al. 1990. Cell 62:991-998; Geisterfer-Lowrance et al. 1990. Cell 61:999-1006), habiéndose identificado más de 100 mutaciones causantes de MCHF (http://www.cardiogenomics.org).Several genetic analyzes have allowed to identify mutations in the MYH7 gene, which encodes the heavy chain of human cardiac beta myosin, associated with the appearance of MCHF (Seidman CE & Seidman JG. 1991. Mol. Biol. Med. 8: 159-166 ; Tanigawa et al . 1990. Cell 62: 991-998; Geisterfer-Lowrance et al . 1990. Cell 61: 999-1006), having identified more than 100 mutations causing MCHF ( http://www.cardiogenomics.org ) .
Actualmente el diagnóstico de la MCHF se basa en los datos clínicos, fundamentalmente el ecocardiograma. Sin embargo, este sistema no permite detectar los casos de muerte súbita que, en general, están provocados por la presencia de una mutación en alguno de los genes implicados. Como es conocido, la MCHF es la primera causa de muerte súbita en personas jóvenes y, dada su prevalencia (1/500), puede estimarse que el número de portadores de alguna de las mutaciones responsables de dicha enfermedad en nuestro país se acerca a las 75.000 personas.Currently the diagnosis of MCHF is based on clinical data, mainly the echocardiogram. But nevertheless, this system does not allow to detect cases of sudden death that, in In general, they are caused by the presence of a mutation in some of the genes involved. As is known, the MCHF is the first cause of sudden death in young people and, given its prevalence (1/500), it can be estimated that the number of carriers of any of the mutations responsible for this disease in our country are Approaches 75,000 people.
El gran tamaño de los genes relacionados hasta la fecha con la aparición de MCH, por ejemplo, el gen MYH7 (28.425 pares de bases) dificulta en gran medida la identificación de mutaciones asociadas con MCH. Esta invención proporciona un método sencillo y rápido para la identificación de algunas mutaciones en el gen MYH7 que pueden ser causa de MCH.The large size of related genes up to the date with the appearance of MCH, for example, the MYH7 gene (28,425 base pairs) makes identification of mutations associated with MCH. This invention provides a method. simple and fast for the identification of some mutations in the MYH7 gene that can cause MCH.
La presente invención se basa en el descubrimiento de una nueva mutación puntual en el gen MYH7 asociada con una mayor predisposición de que el sujeto que la porta desarrolle MCH. La nueva mutación puntual en el gen MYH7 asociada con el desarrollo o la predisposición a desarrollar MCH identificada en esta invención es la mutación Ala797Pro, la cual se definirá detalladamente más adelante.The present invention is based on the discovery of a new point mutation in the associated MYH7 gene with a greater predisposition that the subject who carries it develop MCH. The new point mutation in the associated MYH7 gene with the development or predisposition to develop MCH identified in this invention is the Ala797Pro mutation, which will be defined in detail later.
La identificación de la presencia de dicha mutación en el gen MYH7 en un sujeto con MCH permite tomar medidas preventivas en el caso de los portadores, tales como ejercicio, medicación, consejo genético, etc. En los casos en los que se identifiquen mutaciones consideradas de alto riesgo podrán tomarse medidas adicionales tales como la implantación de un desfibrilador con el fin de evitar la muerte súbita del sujeto portador de tales mutaciones.The identification of the presence of said mutation in the MYH7 gene in a subject with MCH allows taking measurements preventive in the case of carriers, such as exercise, medication, genetic counseling, etc. In cases where identify mutations considered high risk may be taken additional measures such as implantation of a defibrillator in order to avoid the sudden death of the carrier subject of such mutations
Por tanto, en un aspecto, la invención se relaciona con un método para determinar in vitro la predisposición genética de un sujeto a desarrollar MCH que comprende analizar el ácido nucleico contenido en una muestra biológica procedente de dicho sujeto para detectar la presencia o ausencia de una mutación puntual en el gen MYH7 asociada con la MCH, en el que dicha mutación es Ala797Pro.Therefore, in one aspect, the invention relates to a method for determining in vitro the genetic predisposition of a subject to develop MCH which comprises analyzing the nucleic acid contained in a biological sample from said subject to detect the presence or absence of a point mutation in the MYH7 gene associated with MCH, in which said mutation is Ala797Pro.
En otro aspecto, la invención se relaciona con un método de diagnóstico in vitro de MCH que comprende obtener una muestra biológica procedente de un sujeto en donde dicha muestra biológica contiene ácido nucleico y diagnosticar el sujeto de MCH mediante la detección de la presencia de una mutación puntual en el gen MYH7, en el que dicha mutación es Ala797Pro, como indicador de la enfermedad.In another aspect, the invention relates to an in vitro diagnostic method of MCH comprising obtaining a biological sample from a subject in which said biological sample contains nucleic acid and diagnosing the subject of MCH by detecting the presence of a mutation. point in the MYH7 gene, in which said mutation is Ala797Pro, as an indicator of the disease.
En otro aspecto, la invención se relaciona con un método no invasivo para diagnosticar MCH en un sujeto que comprende aislar, a partir de una muestra de sangre procedente de dicho sujeto, un fragmento del ácido nucleico presente en dicha muestra de sangre que comprende, al menos, una de las regiones del gen MYH7 en donde se encuentra la mutación puntual responsable de dicha mutación Ala797Pro, y diagnosticar el sujeto de MCH mediante la detección de la presencia o ausencia de, al menos, dicha mutación, como indicador de la enfermedad.In another aspect, the invention relates to a non-invasive method to diagnose MCH in a subject that comprises isolating, from a blood sample from said subject, a fragment of the nucleic acid present in said blood sample comprising at least one of the regions of the MYH7 gene where the point mutation responsible for said Ala797Pro mutation, and diagnose the subject of MCH by the detection of the presence or absence of at least said mutation, as an indicator of the disease.
La invención proporciona, por tanto, un método para determinar in vitro la predisposición genética de un sujeto a desarrollar MCH, o para diagnosticar un sujeto de MCH, bien sea familiar (MCHF) o esporádica (MCHE), basado en la detección de una nueva mutación puntual en el gen MYH7 relacionada con la aparición de dicha enfermedad. La detección de la mutación en una persona con riesgo o predisposición genética de desarrollar MCH puede realizarse de forma relativamente sencilla y rápida usando el método proporcionado por esta invención.The invention therefore provides a method for determining in vitro the genetic predisposition of a subject to develop MCH, or for diagnosing a subject of MCH, either familiar (MCHF) or sporadic (MCHE), based on the detection of a new point mutation in the MYH7 gene related to the occurrence of said disease. Detection of the mutation in a person at risk or genetic predisposition to develop MCH can be performed relatively easily and quickly using the method provided by this invention.
El método proporcionado por esta invención constituye una herramienta útil de diagnóstico y/o pronóstico que resulta particularmente importante cuando se aplica sobre sujetos asintomáticos de los cuales se sospecha que pueden tener la enfermedad. Los sujetos sintomáticos tienen muchas más probabilidades de ser diagnosticados adecuadamente por un médico. Los sujetos asintomáticos de familias que tengan una historia de MCHF podrían ser diagnosticados usando el método de esta invención lo que permitiría su diagnóstico antes de la aparición de los síntomas. A los sujetos en los que se detecte una mutación responsable de MCHF se les puede aconsejar unas pautas de comportamiento que probablemente prolonguen su vida; por ejemplo, evitar el ejercicio intenso, medicación, etc., y, en su caso, implantarles un desfibrilador.The method provided by this invention It is a useful diagnostic and / or prognostic tool that It is particularly important when applied to subjects asymptomatic of which it is suspected that they may have the disease. Symptomatic subjects have many more Probability of being properly diagnosed by a doctor. Asymptomatic subjects of families who have a history of MCHF could be diagnosed using the method of this invention. which would allow its diagnosis before the appearance of symptom. To subjects in whom a mutation is detected MCHF manager can be advised of guidelines behavior that probably prolongs his life; for example, avoid intense exercise, medication, etc., and, where appropriate, implant a defibrillator.
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Para facilitar la comprensión de la invención objeto de esta solicitud de patente, se expone, a continuación, el significado de algunos términos y expresiones tal como se utilizan en el contexto de la presente invención.To facilitate the understanding of the invention Subject of this patent application, the following is explained meaning of some terms and expressions as they are used in the context of the present invention.
El término "MCH" se refiere a la miocardiopatía hipertrófica e incluye la miocardiopatía hipertrófica familiar (MCHF) y la miocardiopatía hipertrófica esporádica (MCHE).The term "MCH" refers to the hypertrophic cardiomyopathy and includes hypertrophic cardiomyopathy Familial (MCHF) and sporadic hypertrophic cardiomyopathy (MCHE).
El término "predisposición genética" se refiere a la probabilidad de que un sujeto desarrolle una patología determinada (en este caso, MCH); a modo ilustrativo, un sujeto con una predisposición genética elevada tendrá más probabilidades que la media para desarrollar dicha patología, mientras que un sujeto con una predisposición genética reducida tendrá menos probabilidades que la media para desarrollar dicha patología.The term "genetic predisposition" is refers to the probability that a subject develops a pathology determined (in this case, MCH); by way of illustration, a subject with a high genetic predisposition will be more likely than mean to develop said pathology, while a subject with a reduced genetic predisposition will be less likely than the average to develop said pathology.
El término "sujeto" se refiere a un ser humano, de sexo masculino o femenino, de cualquier raza o edad.The term "subject" refers to a being human, male or female, of any race or age.
El término "proteína" se refiere a una cadena molecular de aminoácidos, unidos por enlaces covalentes o no covalentes. El término incluye todas las formas de modificaciones post-traduccionales, por ejemplo glicosilación, fosforilación o acetilación. Los términos "péptido" y "polipéptido" se refieren a cadenas moleculares de aminoácidos que representan un fragmento proteico. Los términos "proteína", "péptido" y "polipéptido", se usan indistintamente.The term "protein" refers to a amino acid molecular chain, linked by covalent bonds or not covalent The term includes all forms of modifications post-translational, for example glycosylation, phosphorylation or acetylation. The terms "peptide" and "polypeptide" refer to molecular chains of amino acids which represent a protein fragment. The terms "protein", "peptide" and "polypeptide", are used interchangeably.
El término "MYH7" se refiere al gen que codifica la proteína denominada "cadena pesada de la beta miosina cardíaca humana". La secuencia de nucleótidos del gen MYH7 es conocida [Jaenicke T et al. 1990. Genomics 8:194-206; NCBI, número de acceso M57965, versión M57965.2 (GI: 24429600)].The term "MYH7" refers to the gene that encodes the protein called "human cardiac beta myosin heavy chain". The nucleotide sequence of the MYH7 gene is known [Jaenicke T et al . 1990. Genomics 8: 194-206; NCBI, accession number M57965, version M57965.2 (GI: 24429600)].
El término "gen" se refiere a una cadena molecular de desoxirribonucleótidos, que codifica una proteína.The term "gene" refers to a chain molecular deoxyribonucleotides, which encodes a protein.
El término "DNA" se refiere al ácido desoxirribonucleico. Una secuencia de DNA es una secuencia de desoxirribonucleótidos.The term "DNA" refers to acid deoxyribonucleic. A DNA sequence is a sequence of deoxyribonucleotides.
El término "cDNA" se refiere a una secuencia de nucleótidos complementaria a una secuencia de mRNA.The term "cDNA" refers to a Nucleotide sequence complementary to an mRNA sequence.
El término "RNA" se refiere al ácido ribonucleico. Una secuencia de RNA es una secuencia de ribonucleótidos.The term "RNA" refers to acid ribonucleic. An RNA sequence is a sequence of ribonucleotides
El término "mRNA" se refiere al ácido ribonucleico mensajero, que es la fracción del RNA total que se traduce a proteínas.The term "mRNA" refers to acid messenger ribonucleic, which is the fraction of total RNA that is Translates to proteins.
El término "secuencia de nucleótidos" o "secuencia nucleotídica" se refiere indistintamente a una secuencia de ribonucleótidos (RNA) o de desoxirribonucleótidos (DNA).The term "nucleotide sequence" or "nucleotide sequence" refers interchangeably to a ribonucleotide (RNA) or deoxyribonucleotide sequence (DNA).
El término "mutación puntual" se refiere a un SNP (single nucleotide polymorphism), es decir, una variante en una única base de una secuencia de nucleótidos determinada.The term "point mutation" refers to a SNP (single nucleotide polymorphism), that is, a variant in a single base of a particular nucleotide sequence.
Para la nomenclatura de las mutaciones puntuales se han seguido las recomendaciones contenidas en las siguientes referencias: (i) denDunnen JT, Antonarakis SE. 2000. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat. 15(1):7-12; y (ii) Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat. 11(1):1-3.For the nomenclature of point mutations the recommendations contained in the following have been followed References: (i) denDunnen JT, Antonarakis SE. 2000. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15 (1): 7-12; and (ii) Antonarakis SE. Recommendations for a nomenclature system for human gene mutations. Nomenclature Working Group. Hum Mutat 11 (1): 1-3.
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La invención proporciona, en general, un método
para evaluar in vitro la predisposición genética de un sujeto
a desarrollar MCH. Dicho método permite identificar aquellos sujetos
que, dentro de un grupo o población de sujetos, presentan una mayor
predisposición genética de desarrollar MCH. Dicho método puede
utilizarse tanto con fines de diagnóstico (método de diagnóstico)
como con fines de pronóstico (método de pronóstico). Un método de
diagnóstico se refiere a un ensayo realizado sobre un sujeto al que
previamente se le ha determinado su pertenencia a un grupo de riesgo
de padecer MCH debido a que presente síntomas o a los resultados de
otro análisis diferente. Un método de pronóstico se refiere a un
método que ayuda a predecir, al menos en parte, el posible
desarrollo de la MCH en un sujeto. A modo ilustrativo, se puede
analizar un sujeto al que previamente no se le ha diagnosticado MCH,
o que carece de síntomas, con el fin de obtener información sobre la
probabilidad de que dicho individuo desarrolle
MCH.The invention generally provides a method for evaluating in vitro the genetic predisposition of a subject to develop MCH. This method allows identifying those subjects that, within a group or population of subjects, have a greater genetic predisposition to develop MCH. This method can be used both for diagnostic purposes (diagnostic method) and for prognostic purposes (prognosis method). A diagnostic method refers to a trial conducted on a subject who has previously been determined to belong to a risk group of MCH due to symptoms or the results of a different analysis. A forecasting method refers to a method that helps predict, at least in part, the possible development of MCH in a subject. By way of illustration, a subject who has not previously been diagnosed with MCH, or who lacks symptoms, can be analyzed in order to obtain information about the likelihood that said individual will develop
MCH
Por tanto, en un primer aspecto, la invención se relaciona con un método para determinar in vitro la predisposición genética de un sujeto a desarrollar MCH que comprende analizar el ácido nucleico contenido en una muestra biológica procedente de dicho sujeto para detectar la presencia o ausencia de una mutación puntual en el gen MYH7 asociada con la MCH, en el que dicha mutación puntual asociada con la MCH, en adelante mutación puntual de la invención, es la mutación Ala797Pro.Therefore, in a first aspect, the invention relates to a method for determining in vitro the genetic predisposition of a subject to develop MCH which comprises analyzing the nucleic acid contained in a biological sample from said subject to detect the presence or absence of a point mutation in the MYH7 gene associated with MCH, wherein said point mutation associated with MCH, hereinafter point mutation of the invention, is the Ala797Pro mutation.
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En una realización particular, la secuencia de nucleótidos del gen MYH7 comprende la SEQ. ID. NO: 1. En este caso, el cambio de nucleótidos relacionado con la mutación puntual de la invención es el siguiente:In a particular embodiment, the sequence of MYH7 gene nucleotides comprise SEQ. ID. NO: 1. In this case, nucleotide change related to the point mutation of the invention is as follows:
- \underline{Mutación}\ underline {Mutation}
- \underline{Cambio \ del \ nucleótido}\ underline {\ nucleotide change}
- Ala797Pro Ala797Pro
- 14131G>C14131G> C
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La presencia de dicha mutación puntual de la invención en el ácido nucleico presente en la muestra biológica procedente del sujeto ensayado es indicativa de la existencia de una mayor predisposición genética de dicho sujeto a desarrollar MCH que la media, o de que dicho sujeto puede ser diagnosticado de MCH o de que dicho sujeto ha desarrollado MCH.The presence of said point mutation of the invention in the nucleic acid present in the biological sample from the subject tested is indicative of the existence of a greater genetic predisposition of said subject to develop MCH than the mean, or that said subject may be diagnosed of MCH or of that said subject has developed MCH.
La puesta en práctica de dicho método comprende la obtención previa de una muestra biológica del sujeto a ensayar y analizar el ácido nucleico contenido en dicha muestra biológica para detectar la presencia o ausencia de la mutación puntual de la invención mediante cualquier método apropiado para detectar tales mutaciones.The implementation of said method comprises prior obtaining of a biological sample of the subject to be tested and analyze the nucleic acid contained in said biological sample to detect the presence or absence of the point mutation of the invention by any appropriate method to detect such mutations
La muestra biológica procedente del sujeto a ensayar puede ser cualquier muestra biológica de dicho sujeto que contenga ácido nucleico. El ácido nucleico presente en dicha muestra biológica puede ser DNA, por ejemplo, DNA genómico (gDNA) o cDNA, o RNA, por ejemplo, mRNA. Para analizar gDNA se puede utilizar prácticamente cualquier muestra biológica que contenga gDNA, por lo que muestras puras de eritrocitos de mamíferos no pueden ser utilizadas. Asimismo, para analizar cDNA o mRNA la muestra biológica debe ser obtenida de células o tejidos que expresen MYH7. A modo ilustrativo, la muestra biológica que contiene ácido nucleico procedente del sujeto a ensayar puede ser una muestra de tejido o de sangre de dicho sujeto. En una realización particular, dicha muestra biológica se obtiene a partir de células nucleadas de sangre periférica del sujeto a ensayar.The biological sample from the subject to to test can be any biological sample of said subject that contain nucleic acid The nucleic acid present in said sample Biological may be DNA, for example, genomic DNA (gDNA) or cDNA, or RNA, for example, mRNA. To analyze gDNA you can use virtually any biological sample containing gDNA, so that pure samples of mammalian erythrocytes cannot be used Also, to analyze cDNA or mRNA the biological sample must be obtained from cells or tissues that express MYH7. By way Illustrative, the biological sample containing nucleic acid from the subject to be tested may be a tissue sample or blood of said subject. In a particular embodiment, said sample Biological is obtained from nucleated blood cells peripheral of the subject to be tested.
El ácido nucleico contenido en la muestra biológica procedente del sujeto a ensayar puede ser aislado por métodos convencionales mediante el empleo de kits comerciales que permiten el aislamiento de ácidos nucleicos. Algunos de los sistemas comerciales para la extracción de ácidos nucleicos están comercializados por diversas compañías, tales como Qiagen y Amersham Biosciences, por citar dos ejemplos. Los protocolos de extracción están bien establecidos en los manuales de instrucciones que se ofrecen junto con los kits de extracción de ácidos nucleicos suministrados por las empresas mencionadas.The nucleic acid contained in the sample biological originating from the subject to be tested can be isolated by conventional methods by using commercial kits that allow the isolation of nucleic acids. Some of the systems commercial for the extraction of nucleic acids are marketed by various companies, such as Qiagen and Amersham Biosciences, to cite two examples. Extraction protocols are well established in the instruction manuals that offer along with nucleic acid extraction kits supplied by the aforementioned companies.
Muchos de los métodos aquí descritos para detectar la presencia o ausencia de una mutación puntual de la invención requieren la amplificación de la secuencia diana del ácido nucleico presente en la muestra biológica procedente del sujeto a ensayar, por ejemplo, la amplificación previa de un fragmento del gen MYH7 que comprende la región en donde se desea estudiar la presencia o ausencia de la mutación puntual de la invención.Many of the methods described here for detect the presence or absence of a timely mutation of the invention require amplification of the acid target sequence nucleic present in the biological sample from the subject to test, for example, the previous amplification of a fragment of the MYH7 gene that comprises the region where you want to study the presence or absence of the point mutation of the invention.
Dicha amplificación puede realizarse por cualquier técnica conocida, preferentemente, mediante la reacción en cadena de la polimerasa (PCR) o cualquiera de sus variantes, por ejemplo, PCR anidada o nested-PCR. Información sobre la realización de PCR y sus variantes puede encontrarse en las patentes norteamericanas US 4.965.188, US 4.800.159, US 4.683.202 y US 4.683.195, así como en las publicaciones de Ausubel et al. eds. Shorts Protocols in Molecular Biology, 3rd edition, Wiley, 1995; e Innis et al., eds., PCR Protocols, Academic Press, 1990.Said amplification can be performed by any known technique, preferably, by polymerase chain reaction (PCR) or any of its variants, for example, nested PCR or nested-PCR. Information on the realization of PCR and its variants can be found in US patents US 4,965,188, US 4,800,159, US 4,683,202 and US 4,683,195, as well as in the publications of Ausubel et al . eds. Shorts Protocols in Molecular Biology, 3rd edition, Wiley, 1995; and Innis et al ., eds., PCR Protocols, Academic Press, 1990.
Otros métodos de amplificación incluyen la reacción en cadena de la ligasa (LCR) (Wu & Wallace. 1989. Genomics 4:560-569; Landegren et al. 1988. Science 241:1077-1080), la amplificación por desplazamiento de banda (G. Walker et al. 1992. Proc. Natl. Acad. Sci. USA 89:392-396; G. Walker et al. 1992. Nucleic Acid Res. 20:1691-1696), la amplificación basada en la transcripción (D. Kwoh et al. 1989. Proc. Natl. Acad. Sci. USA 86:1173-1177), la replicación de secuencia auto-sostenida (3SR) (J. Guatelli et al. 1992. Proc. Natl. Acad. Sci. USA 87:1874-1878), el sistema de la replicasa Q\beta (P. Lizardi et al. 1988. BioTechnology 6:1197-1202), la amplificación basada en la secuencia de ácido nucleico (NASBA) (R. Lewis. 1992. Genetic Engineering News 12(9):1) y la amplificación por la polimerasa del fago phi29 (L. Blanco et al. 1989. J Biol Chem. 264:8935-8940).Other amplification methods include ligase chain reaction (CSF) (Wu & Wallace. 1989. Genomics 4: 560-569; Landegren et al . 1988. Science 241: 1077-1080), bandwidth amplification ( G. Walker et al . 1992. Proc. Natl. Acad. Sci. USA 89: 392-396; G. Walker et al . 1992. Nucleic Acid Res. 20: 1691-1696), transcription-based amplification (D Kwoh et al . 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177), self-sustained sequence replication (3SR) (J. Guatelli et al . 1992. Proc. Natl. Acad. Sci. USA 87: 1874-1878), the Qβ replicase system (P. Lizardi et al . 1988. BioTechnology 6: 1197-1202), nucleic acid sequence based amplification (NASBA) (R. Lewis. 1992. Genetic Engineering News 12 (9): 1) and the amplification by phage polymerase phi29 (L. Blanco et al . 1989. J Biol Chem. 264: 8935-8940).
La presencia o ausencia de la mutación puntual de la invención en el ácido nucleico presente en la muestra biológica procedente del sujeto a ensayar se puede detectar por métodos convencionales, por ejemplo, mediante el empleo de sondas de oligonucleótidos marcadas con un grupo marcador detectable o mediante reacciones enzimáticas específicas.The presence or absence of the point mutation of the invention in the nucleic acid present in the sample biological originating from the subject to be tested can be detected by conventional methods, for example, by using probes of oligonucleotides labeled with a detectable marker group or by specific enzymatic reactions.
En una realización particular, la determinación de la presencia o ausencia de la mutación puntual de la invención en el ácido nucleico presente en la muestra biológica procedente del sujeto a ensayar se realiza mediante el empleo de una sonda de oligonucleótidos específica marcada con un grupo marcador detectable. Se han descrito numerosos ensayos que utilizan sondas marcadas, los cuales pueden ser utilizados en la puesta en práctica de la presente invención (véanse, por ejemplo, las patentes norteamericanas US 4.302.204, US 4.358.535, US 4.563.419 o US 4.994.373). A modo ilustrativo, en una realización concreta, dicha mutación puntual de la invención se detecta mediante el empleo de una sonda de nucleótidos fijada sobre un soporte sólido, en donde dicha sonda de nucleótidos se selecciona entre una sonda de nucleótidos que es completamente complementaria a la secuencia normal de nucleótidos que codifica la cadena pesada de la beta miosina cardíaca humana o a un fragmento de la misma que contiene la región donde se desea estudiar la presencia o ausencia de mutación puntual, y una sonda de nucleótidos que es completamente complementaria a la secuencia de nucleótidos que codifica para una cadena pesada de la beta miosina cardíaca humana mutada (portadora de la mutación a detectar) o a un fragmento de la misma que contiene la región donde se desea estudiar la presencia o ausencia de mutación puntual, y detectar la presencia o ausencia de hibridación.In a particular embodiment, the determination of the presence or absence of the point mutation of the invention in the nucleic acid present in the biological sample from the subject to test is performed by using a probe specific oligonucleotides labeled with a marker group detectable Numerous trials using probes have been described marked, which can be used in the implementation of the present invention (see, for example, patents US 4,302,204, US 4,358,535, US 4,563,419 or US 4,994,373). By way of illustration, in a specific embodiment, said punctual mutation of the invention is detected by the use of a nucleotide probe fixed on a solid support, where said nucleotide probe is selected from a probe of nucleotides that is completely complementary to the sequence normal nucleotide encoding the beta heavy chain human cardiac myosin or a fragment thereof that contains the region where you want to study the presence or absence of mutation punctual, and a nucleotide probe that is completely complementary to the nucleotide sequence that codes for a heavy chain of mutated human cardiac myosin beta (carrier of the mutation to be detected) or to a fragment thereof that contains the region where you want to study the presence or absence of point mutation, and detect the presence or absence of hybridization.
En otra realización particular, la determinación de la presencia o ausencia de la mutación puntual de la invención en el ácido nucleico presente en la muestra biológica procedente del sujeto a ensayar se realiza mediante una reacción enzimática específica para detectar polimorfismos, por ejemplo, mediante digestión con enzimas de restricción y análisis de los fragmentos de restricción (RFLP) (véanse, por ejemplo, las patentes norteamericanas US 5.324.631 y US 5.645.995). A modo ilustrativo, la mutación puntual de la invención puede ser detectada mediante el uso de una enzima de restricción que reconoce específicamente el nucleótido donde reside la mutación puntual asociada a la MCH, verificándose posteriormente la presencia o ausencia de corte de dicha enzima de restricción mediante la separación de los fragmentos de restricción por electroforesis en un gel de agarosa o poliacrilamida (véanse los Ejemplos que acompañan a esta descripción).In another particular embodiment, the determination of the presence or absence of the point mutation of the invention in the nucleic acid present in the biological sample from the subject to test is performed by an enzymatic reaction specific to detect polymorphisms, for example, by restriction enzyme digestion and analysis of fragments of restriction (RFLP) (see, for example, patents US 5,324,631 and US 5,645,995). By way of illustration, the punctual mutation of the invention can be detected by use of a restriction enzyme that specifically recognizes the nucleotide where the point mutation associated with MCH resides, subsequently verifying the presence or absence of cut of said restriction enzyme by separating the fragments restriction by electrophoresis on an agarose gel or polyacrylamide (see the Examples that accompany this description).
Otras técnicas apropiadas para detectar la mutación puntual de la invención incluyen la secuenciación directa de nucleótidos (Ausubel et al. eds. Shorts Protocols in Molecular Biology, 3rd edition, Wiley, 1995; Sambrook et al. 1989. Molecular Cloning 2^{nd} ed. Chap. 13, Cold Spring Harbor Laboratory Press), que puede realizarse por cualquier método apropiado, por ejemplo, mediante el método de secuenciación de didesoxinucleótidos (Sanger), secuenciación química (Maxam & Gilbert) o variantes de los mismos; minisecuenciación directa (US 5.846.710, US 5.888.819, Syvanen et al. 1993, Am. J. Hum. Genet. 52:46-59, Shumaker et al. 1996. Human Mut. 7:346-354, Pastinen et al. 1997. Genome Res. 7:606-614); ensayos de hibridación en array, por ejemplo, el ensayo múltiple de diagnóstico específico de alelo (MASDA) (US 5.834.181; Shuber et al. 1997. Hum. Molec. Genet. 6:337-347); ensayos dependientes de discriminación de alelos basada en hibridación utilizando, por ejemplo, sondas TaqMan (US 5.962.233; Livak et al. 1995. Nature Genet. 9:341-342) o Molecular Beacons (US 5.925.517, Tyagi et al., Nature Biotech, 16:49-53); electroforesis en gel diagonal de fragmentos de restricción dispuestos en placas de microtitulación (MADGE) (Day & Humpohries. 1994. Anal. Biochem. 222:389-395), transferencia de energía de resonancia por fluorescencia (FRET) (US 5.945.283, Chen et al. 1997. Proc. Natl. Acad. Sci. USA 94:10756-10761).Other appropriate techniques for detecting point mutation of the invention include direct nucleotide sequencing (Ausubel et al . Eds. Shorts Protocols in Molecular Biology, 3rd edition, Wiley, 1995; Sambrook et al . 1989. Molecular Cloning 2 nd ed. Chap. 13, Cold Spring Harbor Laboratory Press), which can be performed by any appropriate method, for example, by the method of dideoxynucleotide sequencing (Sanger), chemical sequencing (Maxam & Gilbert) or variants thereof; direct mini-sequencing (US 5,846,710, US 5,888,819, Syvanen et al . 1993, Am. J. Hum. Genet. 52: 46-59, Shumaker et al . 1996. Human Mut. 7: 346-354, Pastinen et al . 1997. Genome Res. 7: 606-614); array hybridization assays, for example, the multiple allele specific diagnostic assay (MASDA) (US 5,834,181; Shuber et al. 1997. Hum. Molec. Genet. 6: 337-347); allegations of hybridization-based allele discrimination using, for example, TaqMan probes (US 5,962,233; Livak et al . 1995. Nature Genet. 9: 341-342) or Molecular Beacons (US 5,925,517, Tyagi et al . , Nature Biotech, 16: 49-53); Diagonal gel electrophoresis of restriction fragments arranged in microtiter plates (MADGE) (Day & Humpohries. 1994. Anal. Biochem. 222: 389-395), fluorescence resonance energy transfer (FRET) (US 5,945,283, Chen et al . 1997. Proc. Natl. Acad. Sci. USA 94: 10756-10761).
La relación de métodos para detectar mutaciones puntuales previamente mencionada es únicamente ilustrativa y no pretende ser exhaustiva. Los expertos en la materia entenderán que cualquier otro método que permita detectar una mutación puntual o un SNP puede ser utilizado para la puesta en práctica de la presente invención y cae dentro del espíritu de la misma.The relationship of methods to detect mutations specific previously mentioned is only illustrative and not It pretends to be exhaustive. Those skilled in the art will understand that any other method that allows to detect a point mutation or a SNP can be used for the implementation of this invention and falls within the spirit of it.
Adicionalmente, si se desea, se puede detectar si el sujeto es heterozigótico u homozigótico para una mutación puntual determinada, es decir, detectar la presencia o ausencia de dichas mutaciones puntuales en ambos cromosomas del sujeto. En general, la presencia de 2 copias de la misma mutación puntual asociada a MCH (es decir, individuos homozigóticos para dicha mutación puntual) puede indicar un mayor riesgo de desarrollar MCH que en un individuo heterozigótico para dicha mutación puntual.Additionally, if desired, it can be detected if the subject is heterozygous or homozygous for a mutation specific point, that is, detect the presence or absence of said point mutations in both chromosomes of the subject. In general, the presence of 2 copies of the same point mutation associated with MCH (i.e. homozygous individuals for said point mutation) may indicate an increased risk of developing MCH than in a heterozygous individual for said point mutation.
La mutación puntual de la invención se ha identificado mediante un ensayo (Ejemplo 1) consistente en la amplificación previa, mediante PCR, de una secuencia diana del gen MYH7 contenido en una muestra de ácido nucleico presente en una muestra de sangre periférica de un sujeto diagnosticado de MCH, seguido de un análisis de polimorfismo conformacional de una hebra (SSCP) y secuenciación directa de los productos de amplificación que mostraron un patrón de movilidad anormal en el gel de SSCP bajo cualquiera de las condiciones ensayadas. La confirmación de la mutación puntual se realizó mediante un análisis RFLP o mediante el diseño de iniciadores degenerados específicos para la mutación puntual en cuestión.The point mutation of the invention has been identified by an assay (Example 1) consisting of the prior amplification, by PCR, of a gene target sequence MYH7 contained in a sample of nucleic acid present in a peripheral blood sample of a subject diagnosed with MCH, followed by a conformational polymorphism analysis of a strand (SSCP) and direct sequencing of amplification products that showed an abnormal mobility pattern in the low SSCP gel Any of the conditions tested. The confirmation of the point mutation was performed by an RFLP analysis or by the Design of degenerate primers specific for mutation timely in question.
En otro aspecto, la invención se relaciona con un método de diagnóstico in vitro de MCH que comprende obtener una muestra biológica procedente de un sujeto en donde dicha muestra biológica contiene ácido nucleico y diagnosticar el sujeto de MCH mediante la detección de la presencia de la mutación puntual de la invención en dicho ácido nucleico, es decir, de la mutación Ala797Pro.In another aspect, the invention relates to an in vitro diagnostic method of MCH which comprises obtaining a biological sample from a subject in which said biological sample contains nucleic acid and diagnosing the MCH subject by detecting the presence of the mutation. point of the invention in said nucleic acid, that is, of the Ala797Pro mutation.
Este método comprende, por tanto, la obtención de una muestra biológica que contiene ácido nucleico del sujeto a ensayar y analizar el ácido nucleico presente en dicha muestra biológica para detectar la presencia o ausencia de la mutación puntual de la invención mediante cualquier método apropiado para detectar tal mutación. La muestra biológica procedente del sujeto debe presentar las mismas características que las mencionadas previamente en relación con el método in vitro para determinar la predisposición genética de un sujeto a desarrollar MCH previamente descrito. Generalmente, antes de proceder a detectar la presencia o ausencia de la mutación puntual de la invención se procede a amplificar la secuencia diana del ácido nucleico presente en la muestra biológica procedente del sujeto a ensayar, es decir, a amplificar un fragmento del gen MYH7 que comprende la región en donde se desea estudiar la presencia o ausencia de la mutación puntual de la invención. Dicha amplificación puede realizarse por cualquiera de las técnicas conocidas mencionadas previamente, por ejemplo, mediante PCR o cualquiera de sus variantes. El análisis del ácido nucleico para detectar la presencia o ausencia de la mutación puntual de la invención asociada con la MCH se puede realizar por cualquiera de las técnicas previamente mencionadas. La existencia de la mutación puntual de la invención en el ácido nucleico presente en la muestra biológica procedente del sujeto ensayado permite diagnosticar al sujeto de MCH.This method therefore comprises obtaining a biological sample containing nucleic acid from the subject to be tested and analyzing the nucleic acid present in said biological sample to detect the presence or absence of the point mutation of the invention by any appropriate method to detect Such a mutation The biological sample from the subject must have the same characteristics as those previously mentioned in relation to the in vitro method to determine the genetic predisposition of a subject to develop MCH previously described. Generally, before detecting the presence or absence of the point mutation of the invention, the target sequence of the nucleic acid present in the biological sample from the subject to be tested is amplified, that is, to amplify a fragment of the MYH7 gene that it comprises the region where it is desired to study the presence or absence of the point mutation of the invention. Said amplification can be carried out by any of the previously mentioned known techniques, for example, by PCR or any of its variants. Nucleic acid analysis to detect the presence or absence of the point mutation of the invention associated with MCH can be performed by any of the previously mentioned techniques. The existence of the point mutation of the invention in the nucleic acid present in the biological sample from the tested subject allows the diagnosis of the MCH subject.
En otro aspecto, la invención se relaciona con un método no invasivo para diagnosticar MCH en un sujeto que comprende aislar, a partir de una muestra de sangre procedente de dicho sujeto, un fragmento del ácido nucleico presente en dicha muestra de sangre que comprende, al menos, la región del gen MYH7 en donde se encuentra la mutación puntual de la invención asociada a MCH, es decir, la mutación Ala797Pro, y diagnosticar el sujeto de MCH mediante la detección de la presencia o ausencia de dicha mutación puntual de la invención en dicho fragmento de ácido nucleico.In another aspect, the invention relates to a non-invasive method for diagnosing MCH in a subject that comprises isolating, from a blood sample from said subject, a fragment of the nucleic acid present in said blood sample comprising, at less, the region of the MYH7 gene where the punctual mutation of the invention associated with MCH is found, that is, the Ala797Pro mutation, and diagnosing the MCH subject by detecting the presence or absence of said punctual mutation of the invention in said nucleic acid fragment.
En una realización particular, dicho método comprende la obtención de una muestra de sangre, por ejemplo, de sangre periférica, del sujeto a ensayar, la amplificación de un fragmento del ácido nucleico presente en dicha muestra de sangre que comprende, al menos, la región del gen MYH7 en donde puede encontrarse la mutación puntual de la invención, analizar la presencia o ausencia de dicha mutación puntual de la invención, y, en su caso, diagnosticar al sujeto de MCH. Tanto la amplificación del ácido nucleico como su análisis para detectar la presencia o ausencia de la mutación puntual de la invención se pueden realizar mediante cualquiera de los métodos previamente descritos. Adicionalmente, si se desea, dicho método comprende la evaluación de los síntomas clínicos de la MCH en el sujeto que está siendo estudiado.In a particular embodiment, said method comprises obtaining a blood sample, for example, from peripheral blood, of the subject to be tested, the amplification of a nucleic acid fragment present in said blood sample that it comprises at least the region of the MYH7 gene where it can find the punctual mutation of the invention, analyze the presence or absence of said point mutation of the invention, and, if necessary, diagnose the subject of MCH. Both amplification of nucleic acid as its analysis to detect the presence or absence of the point mutation of the invention can be performed by any of the previously described methods. Additionally, if desired, said method comprises the evaluation of the clinical symptoms of MCH in the subject being studied.
En otro aspecto, la invención se relaciona con un kit, en particular, un kit útil para determinar la predisposición de un sujeto a desarrollar MCH o para diagnosticar un sujeto de MCH, que comprende, al menos, un reactivo específico para detectar la presencia o ausencia de, al menos, la mutación puntual de la invención, es decir, la mutación Ala797Pro.In another aspect, the invention relates to a kit , in particular, a kit useful for determining the predisposition of a subject to develop MCH or for diagnosing a subject of MCH, comprising at least one specific reagent for detecting the presence or absence of at least the point mutation of the invention, that is, the Ala797Pro mutation.
En una realización particular, dicho reactivo específico para detectar la presencia o ausencia de la mutación puntual de la invención en el gen MYH7 que codifica para la proteína cadena pesada de la beta miosina cardiaca humana, es una sonda de oligonucleótidos que permite distinguir el alelo que contiene una G en la posición 14131 de la SEQ. ID. NO: 1 del alelo que contiene una C en dicha posición.In a particular embodiment, said reagent specific to detect the presence or absence of the mutation point of the invention in the MYH7 gene encoding the protein heavy chain of human cardiac beta myosin, is a probe of oligonucleotides that allows to distinguish the allele that contains a G at position 14131 of the SEQ. ID. NO: 1 of the allele that contains a C in that position.
En otra realización particular, dicho kit comprende, al menos, una enzima de restricción que permite distinguir el alelo que contiene una G en la posición 14131 de la SEQ. ID. NO: 1 del alelo que contiene una C en dicha posición, tal como, por ejemplo, la enzima de restricción Cac8I.In another particular embodiment, said kit it comprises at least one restriction enzyme that allows distinguish the allele that contains a G at position 14131 of the I KNOW THAT. ID. NO: 1 of the allele containing a C in said position, such as, for example, the restriction enzyme Cac8I.
En otra realización particular, el kit proporcionado por esta invención comprende, al menos, un oligonucleótido iniciador útil para la amplificación de un fragmento de ácido nucleico, tal como un fragmento del gen MYH7 que comprende la región en donde se desea estudiar la presencia o ausencia de la mutación puntual de la invención. En una realización particular, dicho oligonucleótido iniciador se selecciona del grupo formado por los oligonucleótidos iniciadores identificados como SEQ. ID. NO: 36, SEQ. ID. NO: 37, y sus mezclas. Dichos oligonucleótidos iniciadores constituyen un aspecto adicional de la presente invención.In another particular embodiment, the kit provided by this invention comprises at least one primer oligonucleotide useful for amplification of a fragment of nucleic acid, such as a fragment of the MYH7 gene comprising the region where you want to study the presence or absence of the punctual mutation of the invention. In a particular embodiment, said initiating oligonucleotide is selected from the group consisting of the initiating oligonucleotides identified as SEQ. ID. NO: 36, I KNOW THAT. ID. NO: 37, and their mixtures. Such initiator oligonucleotides they constitute an additional aspect of the present invention.
Los oligonucleótidos iniciadores identificados como SEQ. ID. NO: 36 (directo) y SEQ. ID. NO: 37 (inverso) permiten amplificar un fragmento correspondiente al exón 21 del gen MYH7 útil para identificar la mutación Ala797Pro (14131G>C).The identified oligonucleotides identified as SEQ. ID. NO: 36 (direct) and SEQ. ID. NO: 37 (reverse) allow amplify a fragment corresponding to exon 21 of the useful MYH7 gene to identify the Ala797Pro mutation (14131G> C).
En otro aspecto, la invención se relaciona con un biochip, en particular, un biochip útil para determinar la predisposición de un sujeto a desarrollar MCH o para diagnosticar un sujeto de MCH, que comprende, al menos, un reactivo específico para detectar la presencia o ausencia de, al menos, la mutación puntual de la invención, es decir, la mutación Ala797Pro en el gen MYH7, soportado sobre un soporte sólido.In another aspect, the invention relates to a biochip , in particular, a biochip useful for determining the predisposition of a subject to develop MCH or for diagnosing a subject of MCH, comprising at least one specific reagent for detecting the presence or absence of at least the point mutation of the invention, that is, the Ala797Pro mutation in the MYH7 gene, supported on a solid support.
En una realización particular, el reactivo específico para detectar la presencia o ausencia de la mutación puntual de la invención, en particular, en la secuencia de nucleótidos del gen MYH7 que codifica la cadena pesada de la beta miosina cardiaca humana, es dicha sonda previamente mencionada en relación con el kit.In a particular embodiment, the reagent specific to detect the presence or absence of the mutation point of the invention, in particular, in the sequence of MYH7 gene nucleotides encoding the beta heavy chain human cardiac myosin, is said probe previously mentioned in relationship with the kit.
En otra realización particular, dicho biochip comprende, al menos, un oligonucleótido iniciador útil para la amplificación de un fragmento de ácido nucleico, tal como un fragmento del gen MYH7 que comprende la región en donde se desea estudiar la presencia o ausencia de la mutación puntual de la invención. En una realización particular, dicho oligonucleótido iniciador se selecciona del grupo formado por los oligonucleótidos iniciadores identificados como SEQ. ID. NO: 36, SEQ. ID. NO: 37, y sus mezclas.In another particular embodiment, said biochip It comprises at least one initiating oligonucleotide useful for the amplification of a nucleic acid fragment, such as a MYH7 gene fragment comprising the region where it is desired study the presence or absence of the point mutation of the invention. In a particular embodiment, said oligonucleotide initiator is selected from the group consisting of oligonucleotides initiators identified as SEQ. ID. NO: 36, SEQ. ID. NO: 37, and their mixtures
Dicho biochip puede contener, además de, al menos uno de dichos oligonucleótidos iniciadores identificados como SEQ. ID. NO: 36, SEQ. ID. NO: 37, uno o más oligonucleótidos iniciadores seleccionados entre los identificados como SEQ. ID. NO: 1- SEQ. ID. NO: 35, y SEQ. ID. NO: 38- SEQ. ID. NO: 85. Estos oligonucleótidos iniciadores amplifican fragmentos incluidos en distintos exones del gen MYH7 (Tabla 1).Said biochip may contain, in addition to, the minus one of said initiating oligonucleotides identified as I KNOW THAT. ID. NO: 36, SEQ. ID. NO: 37, one or more oligonucleotides primers selected from those identified as SEQ. ID. NO: 1- SEQ. ID. NO: 35, and SEQ. ID. NO: 38- SEQ. ID. NO: 85. These initiator oligonucleotides amplify fragments included in different exons of the MYH7 gene (Table 1).
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Los siguientes ejemplos ilustran la invención y no deben ser considerados limitativos del alcance de la misma.The following examples illustrate the invention and They should not be considered as limiting their scope.
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Ejemplo 1Example one
Los miembros de las familias fueron evaluados mediante exploración física, electrocardiograma de 12 derivaciones y ecocardiografía de dos dimensiones. El diagnóstico de MCH se basó en la demostración de la existencia de engrasamiento ventricular y del septo superior al 112% del valor normal en ausencia de hipertensión arterial. Se confeccionó un árbol genealógico de cada paciente y a los familiares de primer grado se les ofreció la posibilidad de acudir a consulta. Se revisaron las historias clínicas de los familiares fallecidos en los casos en los que fue posible. En los casos índice y familiares vivos, previo consentimiento informado se recogieron muestras de sangre periférica para extracción y conservación de ADN.Family members were evaluated by physical examination, 12 lead electrocardiogram and two-dimensional echocardiography. The diagnosis of MCH was based on the demonstration of the existence of ventricular greasing and of septum greater than 112% of normal value in the absence of hypertension arterial. A family tree of each patient was made and a First-degree relatives were offered the possibility of Go to consultation. The medical records of the deceased relatives in cases where it was possible. In the index cases and living relatives, prior informed consent is collected peripheral blood samples for extraction and DNA conservation
Los oligonucleótidos iniciadores utilizados para amplificar distintos fragmentos del gen MYH7 son los indicados en la Tabla 1 (mostrada antes).The initiator oligonucleotides used to amplifying different fragments of the MYH7 gene are those indicated in the Table 1 (shown before).
Se utilizó la reacción en cadena de la polimerasa (PCR) para amplificar las secuencias del gen MYH7 a partir del DNA obtenido de una muestra de sangre periférica procedente de sujetos enfermos (pacientes) de MCH. Los iniciadores usados se relacionan listan en la Tabla 1.The chain reaction of the polymerase (PCR) to amplify MYH7 gene sequences to from the DNA obtained from a peripheral blood sample from sick subjects (patients) of MCH. The initiators used are listed in Table 1.
Las reacciones de amplificación se llevaron a cabo en un volumen final de 50 \muL con 500 ng de DNA en una mezcla de Tris-HCl 20 mM, pH 8,4, KC1 50 mM, MgCl_{2} 1,5 mM, 200 \muM de cada dNTP, 0,2 \muM de cada desoxinucleótido y 1,5 unidades de Taq DNA polimerasa (QIAGEN). Las condiciones de la PCR fueron de un ciclo de desnaturalización a 95ºC durante 10 minutos, seguido de 35 ciclos: desnaturalización a 94ºC durante 1 minuto, hibridación entre 58ºC y 65ºC durante 1 minuto y elongación a 74ºC durante 1 minuto; al final de dichos ciclos se realizó una extensión a 72ºC durante 5 minutos.The amplification reactions were led to conducted in a final volume of 50 µL with 500 ng of DNA in a 20 mM Tris-HCl mixture, pH 8.4, 50 mM KC1, 1.5 mM MgCl2, 200 µM of each dNTP, 0.2 µM of each deoxynucleotide and 1.5 units of Taq DNA polymerase (QIAGEN). The PCR conditions were of a cycle of denaturation at 95 ° C for 10 minutes, followed by 35 cycles: denaturation at 94 ° C for 1 minute, hybridization between 58 ° C and 65 ° C for 1 minute and elongation at 74 ° C for 1 minute; at the end of these cycles made an extension at 72 ° C for 5 minutes.
Se realizó un análisis SSCP de los productos de PCR en un sistema GenePhor (Pharmacia) siguiendo las indicaciones del fabricante. Todas las muestras se corrieron bajo 2 condiciones de temperatura (12ºC y 20ºC) y 2 condiciones de pH (8,3 y 9). Se usaron los iniciadores de la Tabla 1.An SSCP analysis of the products of PCR in a GenePhor system (Pharmacia) following the indications manufacturer. All samples were run under 2 conditions of temperature (12ºC and 20ºC) and 2 pH conditions (8.3 and 9). Be They used the primers in Table 1.
Se realizó secuenciación directa del producto de PCR de todas las muestras que mostraron un patrón de movilidad anormal en el gel de SSCP bajo cualquiera de las condiciones estudiadas. En todos los casos se realizó la secuencia con el iniciador directo e inverso mediante secuenciación automática en un equipo Beckman CQ 2000XL y se siguieron las instrucciones del fabricante. Se usaron los iniciadores que figuran en la Tabla 1.Direct sequencing of the product of PCR of all samples that showed a mobility pattern abnormal in the SSCP gel under any of the conditions studied. In all cases the sequence was performed with the direct and reverse initiator by automatic sequencing in a Beckman CQ 2000XL team and the instructions of the maker. The primers listed in Table 1 were used.
La reacción de secuenciación se llevó a cabo en un termociclador PE Gene Amp System 9700 utilizando los reactivos del kit CEQ2000 Dye terminator Cycle Sequencing con Quick Start de Beckman (Beckman Coulter, Palo Alto, CA, EEUU) y los iniciadores de la Tabla 1. Los fragmentos generados por la reacción de secuenciación se analizaron en un secuenciador automático CEQ 2000XL DNA Analysis System de Beckman. Los cambios observados en la secuencia se confirmaron mediante secuenciación automática de un segundo producto de PCR de la misma muestra. La confirmación posterior en dicho segundo producto de PCR de la misma muestra se llevó a cabo mediante el corte del producto de PCR con una enzima de restricción específica (RFLP) o mediante el diseño de un iniciador degenerado específico para la mutación.The sequencing reaction was carried out in a PE Amp Amp 9700 thermal cycler using reagents of the CEQ2000 Dye terminator Cycle Sequencing kit with Quick Start of Beckman (Beckman Coulter, Palo Alto, CA, USA) and the initiators of Table 1. Fragments generated by the reaction of sequencing were analyzed in an automatic sequencer CEQ 2000XL Beckman's DNA Analysis System. The changes observed in the sequence were confirmed by automatic sequencing of a second PCR product of the same sample. The confirmation subsequent in said second PCR product of the same sample is carried out by cutting the PCR product with an enzyme of specific restriction (RFLP) or by designing an initiator degenerate specific for the mutation.
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
Ejemplo 2Example 2
En la familia FL se encontró la mutación 14131G>C, es decir, un cambio de G (guanina) por C (citosina) en el nucleótido 14131 (según la secuencia del gen MYH7 depositada en la NCBI con el número de acceso M57965, versión M57965.2 (GI: 24429600)] lo que provoca un cambio de alanina (Ala) a prolina (Pro) en el aminoácido 797 de la cadena pesada de la beta miosina cardíaca humana (Ala797Pro).In the FL family the mutation was found 14131G> C, that is, a change from G (guanine) to C (cytosine) in nucleotide 14131 (according to the sequence of the MYH7 gene deposited in The NCBI with access number M57965, version M57965.2 (GI: 24429600)] which causes a change from alanine (Ala) to proline (Pro) at amino acid 797 of the heavy chain of cardiac beta myosin human (Ala797Pro).
\newpage\ newpage
Esta mutación "missense" abole una diana de corte para la enzima de restricción Cac8I que está normalmente presente en el exón 21 lo que proporciona un método independiente para realizar el diagnóstico genético. Se amplificó la secuencia correspondiente al exón 21 del gen MYH7 a partir de DNA genómico obtenido de sangre periférica. Se usaron como iniciadores los oligonucleótidos identificados mediante la SEQ. ID. NO: 36 (directo) y la SEQ. ID. NO: 37 (inverso). El producto de PCR se digirió con Cac8I y posteriormente se sometió a electroforesis en gel de agarosa. La secuencia normal producía 3 fragmentos de 30, 66 y 106 pb respectivamente. La secuencia mutada carecía del sitio de corte para Cac8I, y, por tanto, la mitad del producto de PCR de los individuos portadores tenía el tamaño de 172 pb.This "missense" mutation abolishes a target of cut for the restriction enzyme Cac8I which is normally present in exon 21 which provides an independent method to make the genetic diagnosis. Sequence was amplified corresponding to exon 21 of the MYH7 gene from genomic DNA obtained from peripheral blood. The initiators were used as oligonucleotides identified by SEQ. ID. NO: 36 (direct) and the SEQ. ID. NO: 37 (reverse). The PCR product was digested with Cac8I and subsequently underwent gel electrophoresis of agarose The normal sequence produced 3 fragments of 30, 66 and 106 bp respectively. The mutated sequence lacked the cutting site for Cac8I, and therefore half of the PCR product of the carrier individuals had the size of 172 bp.
<110> UNIVERSIDADE DA CORUÑA<110> UNIVERSIDADE DA CORUÑA
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<120> MÉTODO PARA DIAGNOSTICAR O DETERMINAR LA PREDISPOSICIÓN GENÉTICA A DESARROLLAR MIOCARDIOPATÍA HIPERTRÓFICA<120> METHOD FOR DIAGNOSING OR DETERMINE THE GENETIC PREDISPOSITION TO BE DEVELOPED MYOCARDIOPATHY HYPERTRÓFICA
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<160> 85<160> 85
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 1<210> 1
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 28452<211> 28452
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Homo sapiens <213> Homo sapiens
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 1<400> 1
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 2<210> 2
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 3 del gen MYH7 en combinación con la SEQ. ID. NO: 3<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 3 of the gene MYH7 in combination with the SEQ. ID. NO: 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 2<400> 2
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipggcagccagc ttctgctcac t
\hfill21
\ hskip-.1em \ dddseqskipggcagccagc ttctgctcac t
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 3<210> 3
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 3 del gen MYH7 en combinación con la SEQ. ID. NO: 2<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 3 of the gene MYH7 in combination with the SEQ. ID. NO: 2
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 3<400> 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgactctcaca tcagcctgac acc
\hfill23
\ hskip-.1em \ dddseqskipgactctcaca tcagcctgac acc
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 4<210> 4
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 4 del gen MYH7 en combinación con la SEQ. ID. NO: 5<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 4 of the gene MYH7 in combination with the SEQ. ID. NO: 5
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 4<400> 4
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcactcacca actcctaacc
\hfill20
\ hskip-.1em \ dddseqskiptcactcacca actcctaacc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 5<210> 5
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 4 del gen MYH7 en combinación con la SEQ. ID. NO: 4<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 4 of the gene MYH7 in combination with the SEQ. ID. NO: 4
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 5<400> 5
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgggtggacat ggatggagca
\hfill20
\ hskip-.1em \ dddseqskipgggtggacat ggatggagca
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 6<210> 6
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 5 del gen MYH7 en combinación con la SEQ. ID. NO: 7<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 5 of the gene MYH7 in combination with the SEQ. ID. NO: 7
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 6<400> 6
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgcaactggca agtcactgct c
\hfill21
\ hskip-.1em \ dddseqskipgcaactggca agtcactgct c
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 7<210> 7
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 5 del gen MYH7 en combinación con la SEQ. ID. NO: 6<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 5 of the gene MYH7 in combination with the SEQ. ID. NO 6
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 7<400> 7
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipccagttccct tcaggaagac ctt
\hfill23
\ hskip-.1em \ dddseqskipccagttccct tcaggaagac ctt
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 8<210> 8
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 6 del gen MYH7 en combinación con la SEQ. ID. NO: 9<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 6 of the gene MYH7 in combination with the SEQ. ID. NO: 9
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 8<400> 8
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgagaagcccc acgagagcat
\hfill20
\ hskip-.1em \ dddseqskipgagaagcccc acgagagcat
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 9<210> 9
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 6 del gen MYH7 en combinación con la SEQ. ID. NO: 8<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 6 of the gene MYH7 in combination with the SEQ. ID. NO: 8
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 9<400> 9
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptggaggctgg gatcagggag
\hfill20
\ hskip-.1em \ dddseqskiptggaggctgg gatcagggag
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 10<210> 10
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 7 del gen MYH7 en combinación con la SEQ. ID. NO: 11<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 7 of the gene MYH7 in combination with the SEQ. ID. NO: 11
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 10<400> 10
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctgatttgag gcttgctggt
\hfill20
\ hskip-.1em \ dddseqskipctgatttgag gcttgctggt
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 11<210> 11
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 7 del gen MYH7 en combinación con la SEQ. ID. NO: 10<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 7 of the gene MYH7 in combination with the SEQ. ID. NO: 10
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 11<400> 11
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaagaaggagg caggtgagag
\hfill20
\ hskip-.1em \ dddseqskipaagaaggagg caggtgagag
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 12<210> 12
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 8+9 del gen MYH7 en combinación con la SEQ. ID. NO: 13<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 8 + 9 of the gene MYH7 in combination with the SEQ. ID. NO: 13
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 12<400> 12
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctctcacctg cctccttctt gg
\hfill22
\ hskip-.1em \ dddseqskipctctcacctg cctccttctt gg
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 13<210> 13
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 8+9 del gen MYH7 en combinación con la SEQ. ID. NO: 12<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 8 + 9 of the gene MYH7 in combination with the SEQ. ID. NO: 12
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 13<400> 13
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgctgagccta gcagattcat gg
\hfill22
\ hskip-.1em \ dddseqskipgctgagccta gcagattcat gg
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 14<210> 14
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 10 del gen MYH7 en combinación con la SEQ. ID. NO: 15<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 10 of the gene MYH7 in combination with the SEQ. ID. NO: 15
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 14<400> 14
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgtgcccaaac cctaactttt ct
\hfill22
\ hskip-.1em \ dddseqskipgtgcccaaac cctaactttt ct
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 15<210> 15
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 10 del gen MYH7 en combinación con la SEQ. ID. NO: 14<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 10 of the gene MYH7 in combination with the SEQ. ID. NO: 14
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 15<400> 15
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipatagttggtc tcagtcggtg gc
\hfill22
\ hskip-.1em \ dddseqskipatagttggtc tcagtcggtg gc
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 16<210> 16
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 11 del gen MYH7 en combinación con la SEQ. ID. NO: 17<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 11 of the gene MYH7 in combination with the SEQ. ID. NO: 17
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 16<400> 16
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcttgtgtccc accctaacca tgt
\hfill23
\ hskip-.1em \ dddseqskipcttgtgtccc accctaacca tgt
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 17<210> 17
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 11 del gen MYH7 en combinación con la SEQ. ID. NO: 16<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 11 of the gene MYH7 in combination with the SEQ. ID. NO: 16
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 17<400> 17
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptttgcccctc actgccaatc ctc
\hfill23
\ hskip-.1em \ dddseqskiptttgcccctc actgccaatc ctc
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 18<210> 18
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 12 del gen MYH7 en combinación con la SEQ. ID. NO: 19<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 12 of the gene MYH7 in combination with the SEQ. ID. NO: 19
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 18<400> 18
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptttctggggt ccgccaatat gg
\hfill22
\ hskip-.1em \ dddseqskiptttctggggt ccgccaatat gg
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 19<210> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 12 del gen MYH7 en combinación con la SEQ. ID. NO: 18<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 12 of the gene MYH7 in combination with the SEQ. ID. NO: 18
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 19<400> 19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagctgcccca agaatcctgc ct
\hfill22
\ hskip-.1em \ dddseqskipagctgcccca agaatcctgc ct
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 20<210> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 13 del gen MYH7 en combinación con la SEQ. ID. NO: 21<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 13 of the gene MYH7 in combination with the SEQ. ID. NO: 21
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 20<400> 20
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctcttaccaa ctttgctgtt gcc
\hfill23
\ hskip-.1em \ dddseqskipctcttaccaa ctttgctgtt gcc
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 21<210> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 13 del gen MYH7 en combinación con la SEQ. ID. NO: 20<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 13 of the gene MYH7 in combination with the SEQ. ID. NO: 20
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 21<400> 21
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcctgcccacc cattatcatc tg
\hfill22
\ hskip-.1em \ dddseqskipcctgcccacc cattatcatc tg
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 22<210> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 25<211> 25
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 14 del gen MYH7 en combinación con la SEQ. ID. NO: 23<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 14 of the gene MYH7 in combination with the SEQ. ID. NO: 23
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 22<400> 22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcttcccaaca accctgctca atatg
\hfill25
\ hskip-.1em \ dddseqskipcttcccaaca accctgctca atatg
\ hfill25
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 23<210> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 14 del gen MYH7 en combinación con la SEQ. ID. NO: 22<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 14 of the gene MYH7 in combination with the SEQ. ID. NO: 22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 23<400> 23
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgtgattgttc tcccactccc agg
\hfill23
\ hskip-.1em \ dddseqskipgtgattgttc tcccactccc agg
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 24<210> 24
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 24<211> 24
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 15 del gen MYH7 en combinación con la SEQ. ID. NO: 25<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 15 of the gene MYH7 in combination with the SEQ. ID. NO: 25
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 24<400> 24
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptttctgactg ctcccacccc tcat
\hfill24
\ hskip-.1em \ dddseqskiptttctgactg ctcccacccc tcat
\ hfill24
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 25<210> 25
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 15 del gen MYH7 en combinación con la SEQ. ID. NO: 24<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 15 of the gene MYH7 in combination with the SEQ. ID. NO: 24
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 25<400> 25
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgggagaattc aggtggtaag gc
\hfill22
\ hskip-.1em \ dddseqskipgggagaattc aggtggtaag gc
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 26<210> 26
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 16 del gen MYH7 en combinación con la SEQ. ID. NO: 27<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 16 of the gene MYH7 in combination with the SEQ. ID. NO: 27
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 26<400> 26
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipttgaccatag agcagaatcc
\hfill20
\ hskip-.1em \ dddseqskipttgaccatag agcagaatcc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 27<210> 27
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 16 del gen MYH7 en combinación con la SEQ. ID. NO: 26<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 16 of the gene MYH7 in combination with the SEQ. ID. NO: 26
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 27<400> 27
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctcagaacct tggcagaatc
\hfill20
\ hskip-.1em \ dddseqskipctcagaacct tggcagaatc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 28<210> 28
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 17 del gen MYH7 en combinación con la SEQ. ID. NO: 29<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 17 of the gene MYH7 in combination with the SEQ. ID. NO: 29
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 28<400> 28
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcttactcac accctacctc
\hfill20
\ hskip-.1em \ dddseqskiptcttactcac accctacctc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 29<210> 29
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 19<211> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 17 del gen MYH7 en combinación con la SEQ. ID. NO: 28<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 17 of the gene MYH7 in combination with the SEQ. ID. NO: 28
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 29<400> 29
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgggctgggtg gggttgggc
\hfill19
\ hskip-.1em \ dddseqskipgggctgggtg gggttgggc
\ hfill19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 30<210> 30
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 18 del gen HYH7 en combinación con la SEQ. ID. NO: 31<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 18 of the gene HYH7 in combination with the SEQ. ID. NO: 31
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 30<400> 30
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcttcctgcat ctctttctgg ca
\hfill22
\ hskip-.1em \ dddseqskipcttcctgcat ctctttctgg ca
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 31<210> 31
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 18 del gen MYH7 en combinación con la SEQ. ID. NO: 30<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 18 of the gene MYH7 in combination with the SEQ. ID. NO: 30
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 31<400> 31
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagcagtgggt tggccctagt tt
\hfill22
\ hskip-.1em \ dddseqskipagcagtgggt tggccctagt tt
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 32<210> 32
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 24<211> 24
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 19 del gen MYH7 en combinación con la SEQ. ID. NO: 33<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 19 of the gene MYH7 in combination with the SEQ. ID. NO: 33
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 32<400> 32
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipccagttctca cagactcctc ctac
\hfill24
\ hskip-.1em \ dddseqskipccagttctca cagactcctc ctac
\ hfill24
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 33<210> 33
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 19 del gen MYH7 en combinación con la SEQ. ID. NO: 32<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 19 of the gene MYH7 in combination with the SEQ. ID. NO: 32
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 33<400> 33
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipccctgttcta tgagcttctg gtg
\hfill23
\ hskip-.1em \ dddseqskipccctgttcta tgagcttctg gtg
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 34<210> 34
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 20 del gen MYH7 en combinación con la SEQ. ID. NO: 35<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 20 of the gene MYH7 in combination with the SEQ. ID. NO: 35
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 34<400> 34
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipggatctgcag gtgaccctag aat
\hfill23
\ hskip-.1em \ dddseqskipggatctgcag gtgaccctag aat
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 35<210> 35
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 20 del gen MYH7 en combinación con la SEQ. ID. NO: 34<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 20 of the gene MYH7 in combination with the SEQ. ID. NO: 34
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 35<400> 35
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipacaacaggaa aagcatcaga gg
\hfill22
\ hskip-.1em \ dddseqskipacaacaggaa aagcatcaga gg
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 36<210> 36
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 21 del gen MYH7 en combinación con la SEQ. ID. NO: 37<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 21 of the gene MYH7 in combination with the SEQ. ID. NO: 37
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 36<400> 36
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptccctagtca tggccaacac a
\hfill21
\ hskip-.1em \ dddseqskiptccctagtca tggccaacac a
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 37<210> 37
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 21 del gen MYH7 en combinación con la SEQ. ID. NO: 36<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 21 of the gene MYH7 in combination with the SEQ. ID. NO: 36
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 37<400> 37
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagcgggaaac ctcctcttga g
\hfill21
\ hskip-.1em \ dddseqskipagcgggaaac ctcctcttga g
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 38<210> 38
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 22 A del gen MYH7 en combinación con la SEQ. ID. NO: 39<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 22 A of the MYH7 gene in combination with SEQ. ID. NO: 39
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 38<400> 38
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcaggacctc aggtaggaag gag
\hfill23
\ hskip-.1em \ dddseqskiptcaggacctc aggtaggaag gag
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 39<210> 39
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 22 A del gen MYH7 en combinación con la SEQ. ID. NO: 38<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 22 A of the MYH7 gene in combination with SEQ. ID. NO: 38
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 39<400> 39
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctctttgagg cgtgtgaact cc
\hfill22
\ hskip-.1em \ dddseqskipctctttgagg cgtgtgaact cc
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 40<210> 40
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 22 B del gen MYH7 en combinación con la SEQ. ID. NO: 41<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 22 B of the MYH7 gene in combination with SEQ. ID. NO: 41
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 40<400> 40
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgctgaagagt gcagaaagag ag
\hfill22
\ hskip-.1em \ dddseqskipgctgaagagt gcagaaagag ag
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 41<210> 41
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 22 B del gen MYH7 en combinación con la SEQ. ID. NO: 40<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 22 B of the MYH7 gene in combination with SEQ. ID. NO: 40
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 41<400> 41
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgtgggaagt gaaggcagag
\hfill20
\ hskip-.1em \ dddseqskiptgtgggaagt gaaggcagag
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 42<210> 42
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 23 A del gen MYH7 en combinación con la SEQ. ID. NO: 43<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 23 A of the MYH7 gene in combination with SEQ. ID. NO: 43
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 42<400> 42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaagaatgagg accttacccc ctg
\hfill23
\ hskip-.1em \ dddseqskipaagaatgagg accttacccc ctg
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 43<210> 43
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 23 A del gen MYH7 en combinación con la SEQ. ID. NO: 42<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 23 A of the MYH7 gene in combination with SEQ. ID. NO: 42
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 43<400> 43
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgctctgagca ctcatcttcc aac
\hfill23
\ hskip-.1em \ dddseqskipgctctgagca ctcatcttcc aac
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 44<210> 44
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 23 B del gen MYH7 en combinación con la SEQ. ID. NO: 45<223> Direct initiator oligonucleotide used for the amplification of a fragment of exon 23 B of the MYH7 gene in combination with SEQ. ID. NO: 45
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 44<400> 44
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipggctaaggtg aaggagatga ac
\hfill22
\ hskip-.1em \ dddseqskipggctaaggtg aaggagatga ac
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 45<210> 45
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 23 B del gen MYH7 en combinación con la SEQ. ID. NO: 44<223> Reverse initiator oligonucleotide used for the amplification of a fragment of exon 23 B of the MYH7 gene in combination with SEQ. ID. NO: 44
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 45<400> 45
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptctgagagtc ctgatgagac cc
\hfill22
\ hskip-.1em \ dddseqskiptctgagagtc ctgatgagac cc
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 46<210> 46
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 23<211> 23
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 24 del gen MYH7 en combinación con la SEQ. ID. NO: 47<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 24 of the gene MYH7 in combination with the SEQ. ID. NO: 47
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 46<400> 46
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipccctaaagga gatgggattc ttg
\hfill23
\ hskip-.1em \ dddseqskipccctaaagga gatgggattc ttg
\ hfill2. 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 47<210> 47
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 24 del gen MYH7 en combinación con la SEQ. ID. NO: 46<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 24 of the gene MYH7 in combination with the SEQ. ID. NO: 46
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 47<400> 47
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptctaggcccc acaactctca at
\hfill22
\ hskip-.1em \ dddseqskiptctaggcccc acaactctca at
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 48<210> 48
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación<223> Direct initiator oligonucleotide used for amplification
de un fragmento del exón 25 del gen MYH7 en combinación con la SEQ. ID. NO: 49of a fragment of exon 25 of the MYH7 gene in combination with the SEQ. ID. NO: 49
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 48<400> 48
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagtcaggatg ctttgcctca
\hfill20
\ hskip-.1em \ dddseqskipagtcaggatg ctttgcctca
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 49<210> 49
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 25 del gen MYH7 en combinación con la SEQ. ID. NO: 48<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 25 of the gene MYH7 in combination with the SEQ. ID. NO: 48
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 49<400> 49
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipttgtactgtt atgggctggg
\hfill20
\ hskip-.1em \ dddseqskipttgtactgtt atgggctggg
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 50<210> 50
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 26 del gen MYH7 en combinación con la SEQ. ID. NO: 51<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 26 of the gene MYH7 in combination with the SEQ. ID. NO: 51
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 50<400> 50
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipattttcacca ctggtggacc
\hfill20
\ hskip-.1em \ dddseqskipattttcacca ctggtggacc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 51<210> 51
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 26 del gen MYH7 en combinación con la SEQ. ID. NO: 50<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 26 of the gene MYH7 in combination with the SEQ. ID. NO: 50
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 51<400> 51
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaatgctgtga acaggacacc
\hfill20
\ hskip-.1em \ dddseqskipaatgctgtga acaggacacc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 52<210> 52
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 19<211> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 27 A del gen MYH7 en combinación con la SEQ. ID. NO: 53<223> Reverse initiator oligonucleotide used for the amplification of a fragment of exon 27 A of the MYH7 gene in combination with SEQ. ID. NO: 53
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 52<400> 52
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipattccagtgg aggggtcca
\hfill19
\ hskip-.1em \ dddseqskipattccagtgg aggggtcca
\ hfill19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 53<210> 53
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 19<211> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 27 A del gen MYH7 en combinación con la SEQ. ID. NO: 52<223> Reverse initiator oligonucleotide used for the amplification of a fragment of exon 27 A of the MYH7 gene in combination with SEQ. ID. NO: 52
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 53<400> 53
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcgccgcatct tctggaact
\hfill19
\ hskip-.1em \ dddseqskipcgccgcatct tctggaact
\ hfill19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 54<210> 54
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 19<211> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 27 B del gen MYH7 en combinación con la SEQ. ID. NO: 55<223> Direct initiator oligonucleotide used for the amplification of a fragment of exon 27 B of the MYH7 gene in combination with SEQ. ID. NO: 55
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 54<400> 54
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipagttccagaa gatgcggcg
\hfill19
\ hskip-.1em \ dddseqskipagttccagaa gatgcggcg
\ hfill19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 55<210> 55
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 27 B del gen MYH7 en combinación con la SEQ. ID. NO: 54<223> Reverse initiator oligonucleotide used for the amplification of a fragment of exon 27 B of the MYH7 gene in combination with SEQ. ID. NO: 54
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 55<400> 55
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgggaggagg aagttggagg a
\hfill21
\ hskip-.1em \ dddseqskiptgggaggagg aagttggagg to
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 56<210> 56
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 28 del gen MYH7 en combinación con la SEQ. ID. NO: 57<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 28 of the gene MYH7 in combination with the SEQ. ID. NO: 57
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 56<400> 56
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgcacctctta cacccctca
\hfill20
\ hskip-.1em \ dddseqskipgcacctctta cacccctca
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 57<210> 57
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 28 del gen MYH7 en combinación con la SEQ. ID. NO: 56<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 28 of the gene MYH7 in combination with the SEQ. ID. NO: 56
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 57<400> 57
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgtgcgtgta ttggcttgtg
\hfill20
\ hskip-.1em \ dddseqskiptgtgcgtgta ttggcttgtg
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 58<210> 58
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 29 del gen MYH7 en combinación con la SEQ. ID. NO: 59<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 29 of the gene MYH7 in combination with the SEQ. ID. NO: 59
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 58<400> 58
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaggtggggat agagaggagt
\hfill20
\ hskip-.1em \ dddseqskipaggtggggat agagaggagt
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 59<210> 59
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 29 del gen MYH7 en combinación con la SEQ. ID. NO: 58<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 29 of the gene MYH7 in combination with the SEQ. ID. NO: 58
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 59<400> 59
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgatgcaaggc tagtcagtgt g
\hfill21
\ hskip-.1em \ dddseqskipgatgcaaggc tagtcagtgt g
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 60<210> 60
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 30 del gen MYH7 en combinación con la SEQ. ID. NO: 61<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 30 of the gene MYH7 in combination with the SEQ. ID. NO: 61
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 60<400> 60
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipatggagaaag ctgaacccac
\hfill20
\ hskip-.1em \ dddseqskipatggagaaag ctgaacccac
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 61<210> 61
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 19<211> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 30 del gen MYH7 en combinación con la SEQ. ID. NO: 60<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 30 of the gene MYH7 in combination with the SEQ. ID. NO: 60
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 61<400> 61
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctgagtcctg cctgcaaag
\hfill19
\ hskip-.1em \ dddseqskipctgagtcctg cctgcaaag
\ hfill19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 62<210> 62
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 31 del gen MYH7 en combinación con la SEQ. ID. NO: 63<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 31 of the gene MYH7 in combination with the SEQ. ID. NO: 63
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 62<400> 62
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgtttcctct tgtccccatc
\hfill20
\ hskip-.1em \ dddseqskiptgtttcctct tgtccccatc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 63<210> 63
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 31 del gen MYH7 en combinación con la SEQ. ID. NO: 62<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 31 of the gene MYH7 in combination with the SEQ. ID. NO: 62
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 63<400> 63
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctctcactga acccctcatg
\hfill20
\ hskip-.1em \ dddseqskipctctcactga acccctcatg
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 64<210> 64
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 32 del gen MYH7 en combinación con la SEQ. ID. NO: 65<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 32 of the gene MYH7 in combination with the SEQ. ID. NO: 65
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 64<400> 64
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctgagacaga ccctggacat
\hfill20
\ hskip-.1em \ dddseqskipctgagacaga ccctggacat
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 65<210> 65
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 32 del gen MYH7 en combinación con la SEQ. ID. NO: 64<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 32 of the gene MYH7 in combination with the SEQ. ID. NO: 64
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 65<400> 65
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgcaccatatg ggaacactgc
\hfill20
\ hskip-.1em \ dddseqskipgcaccatatg ggaacactgc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 66<210> 66
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 33 del gen MYH7 en combinación con la SEQ. ID. NO: 67<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 33 of the gene MYH7 in combination with the SEQ. ID. NO: 67
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 66<400> 66
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptggacctcag cagccctcaa a
\hfill21
\ hskip-.1em \ dddseqskiptggacctcag cagccctcaa a
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 67<210> 67
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 33 del gen MYH7 en combinación con la SEQ. ID. NO: 66<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 33 of the gene MYH7 in combination with the SEQ. ID. NO: 66
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 67<400> 67
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaccaaaagcc tggagctcag
\hfill20
\ hskip-.1em \ dddseqskipaccaaaagcc tggagctcag
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 68<210> 68
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 34 A del gen MYH7 en combinación con la SEQ. ID. NO: 69<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 34 A of the MYH7 gene in combination with SEQ. ID. NO: 69
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 68<400> 68
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipatccatgatt agtgagcagg cc
\hfill22
\ hskip-.1em \ dddseqskipatccatgatt agtgagcagg cc
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 69<210> 69
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 34 A del gen MYH7 en combinación con la SEQ. ID. NO: 68<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 34 A of the MYH7 gene in combination with SEQ. ID. NO: 68
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 69<400> 69
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcttcttctt caccctcagg g
\hfill21
\ hskip-.1em \ dddseqskiptcttcttctt caccctcagg g
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 70<210> 70
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 34 B del gen MYH7 en combinación con la SEQ. ID. NO: 71<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 34 B of the MYH7 gene in combination with SEQ. ID. NO: 71
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 70<400> 70
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipccctgagggt gaagaagaag a
\hfill21
\ hskip-.1em \ dddseqskipccctgagggt gaagaagaag a
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 71<210> 71
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 34 B del gen MYH7 en combinación con la SEQ. ID. NO: 70<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 34 B of the MYH7 gene in combination with SEQ. ID. NO: 70
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 71<400> 71
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptcaagacact actgcttacg cc
\hfill22
\ hskip-.1em \ dddseqskiptcaagacact actgcttacg cc
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 72<210> 72
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 35 del gen MYH7 en combinación con la SEQ. ID. NO: 73<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 35 of the gene MYH7 in combination with the SEQ. ID. NO: 73
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 72<400> 72
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptagtgaaggg aaccgaggct
\hfill20
\ hskip-.1em \ dddseqskiptagtgaaggg aaccgaggct
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 73<210> 73
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 35 del gen MYH7 en combinación con la SEQ. ID. NO: 72<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 35 of the gene MYH7 in combination with the SEQ. ID. NO: 72
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 73<400> 73
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipgctcccttca ggaatgagca
\hfill20
\ hskip-.1em \ dddseqskipgctcccttca ggaatgagca
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 74<210> 74
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 36 del gen MYH7 en combinación con la SEQ. ID. NO: 75<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 36 of the gene MYH7 in combination with the SEQ. ID. NO: 75
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 74<400> 74
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcaaggcttga gagctatgca
\hfill20
\ hskip-.1em \ dddseqskipcaaggcttga gagctatgca
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 75<210> 75
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 36 del gen MYH7 en combinación con la SEQ. ID. NO: 74<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 36 of the gene MYH7 in combination with the SEQ. ID. NO: 74
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 75<400> 75
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctggtcaagt cctcacacac t
\hfill21
\ hskip-.1em \ dddseqskipctggtcaagt cctcacacac t
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 76<210> 76
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 19<211> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 37 A del gen MYH7 en combinación con la SEQ. ID. NO: 77<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 37 A of the MYH7 gene in combination with SEQ. ID. NO: 77
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 76<400> 76
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgggcagcaa gtgtgtgag
\hfill19
\ hskip-.1em \ dddseqskiptgggcagcaa gtgtgtgag
\ hfill19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 77<210> 77
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 19<211> 19
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 37 A del gen MYH7 en combinación con la SEQ. ID. NO: 76<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 37 A of the MYH7 gene in combination with SEQ. ID. NO: 76
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 77<400> 77
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcttctgcagc tgcttcttg
\hfill19
\ hskip-.1em \ dddseqskipcttctgcagc tgcttcttg
\ hfill19
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 78<210> 78
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 37 B del gen MYH7 en combinación con la SEQ. ID. NO: 79<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 37 B of the MYH7 gene in combination with SEQ. ID. NO: 79
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 78<400> 78
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcaagaagcag ctgcagaagc
\hfill20
\ hskip-.1em \ dddseqskipcaagaagcag ctgcagaagc
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 79<210> 79
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 37 B del gen MYH7 en combinación con la SEQ. ID. NO: 78<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 37 B of the MYH7 gene in combination with SEQ. ID. NO: 78
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 79<400> 79
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipcctcagctgg ttgtcactgt
\hfill20
\ hskip-.1em \ dddseqskipcctcagctgg ttgtcactgt
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 80<210> 80
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 22<211> 22
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 38 del gen MYH7 en combinación con la SEQ. ID. NO: 81<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 38 of the gene MYH7 in combination with the SEQ. ID. NO: 81
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 80<400> 80
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipctatgactgt gccatcttca cc
\hfill22
\ hskip-.1em \ dddseqskipctatgactgt gccatcttca cc
\ hfill22
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 81<210> 81
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 38 del gen MYH7 en combinación con la SEQ. ID. NO: 80<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 38 of the gene MYH7 in combination with the SEQ. ID. NO: 80
\newpage\ newpage
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 81<400> 81
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipggttctcaga ctcctggctt
\hfill20
\ hskip-.1em \ dddseqskipggttctcaga ctcctggctt
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 82<210> 82
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador directo utilizado para la amplificación de un fragmento del exón 3 9 del gen MYH7 en combinación con la SEQ. ID. NO: 83<223> Direct initiator oligonucleotide used for amplification of a fragment of exon 3 9 of the gene MYH7 in combination with the SEQ. ID. NO: 83
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 82<400> 82
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaagccaggag tctgagaacc c
\hfill21
\ hskip-.1em \ dddseqskipaagccaggag tctgagaacc c
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 83<210> 83
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 39 del gen MYH7 en combinación con la SEQ. ID. NO: 82<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 39 of the gene MYH7 in combination with the SEQ. ID. NO: 82
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 83<400> 83
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptgtctgggta tgcctgctgt
\hfill20
\ hskip-.1em \ dddseqskiptgtctgggta tgcctgctgt
\ hfilltwenty
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 84<210> 84
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 40 del gen MYH7 en combinación con la SEQ. ID. NO: 85<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 40 of the gene MYH7 in combination with the SEQ. ID. NO: 85
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 84<400> 84
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskipaccatcagac ccctctcacc t
\hfill21
\ hskip-.1em \ dddseqskipaccatcagac ccctctcacc t
\ hfilltwenty-one
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 85<210> 85
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 20<211> 20
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> ADN<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> Secuencia artificial<213> Artificial sequence
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<223> Oligonucleótido iniciador inverso utilizado para la amplificación de un fragmento del exón 40 del gen MYH7 en combinación con la SEQ. ID. NO: 84<223> Reverse initiator oligonucleotide used for amplification of a fragment of exon 40 of the gene MYH7 in combination with the SEQ. ID. NO: 84
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 85<400> 85
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
\hskip-.1em\dddseqskiptattctgctt cctcccaagg
\hfill20
\ hskip-.1em \ dddseqskiptattctgctt cctcccaagg
\ hfilltwenty
Claims (31)
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
- \underline{Mutación}\ underline {Mutation}
- \underline{Cambio \ del \ nucleótido}\ underline {\ nucleotide change}
- Ala797Pro Ala797Pro
- 14131G>C14131G> C
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
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| ES200401428A ES2317713B1 (en) | 2004-06-07 | 2004-06-07 | METHOD FOR DIAGNOSING OR DETERMINING THE GENETIC PREDISPOSITION TO DEVELOP HYPERTROPHIC MIOCARDIOPATIA. |
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| ES200401428A Expired - Fee Related ES2317713B1 (en) | 2004-06-07 | 2004-06-07 | METHOD FOR DIAGNOSING OR DETERMINING THE GENETIC PREDISPOSITION TO DEVELOP HYPERTROPHIC MIOCARDIOPATIA. |
| ES200803452A Active ES2340459B1 (en) | 2004-06-07 | 2008-12-01 | METHOD FOR DIAGNOSING OR DETERMINING THE GENETIC PREDISPOSITION TO DEVELOP HYPERTROPHIC MIOCARDIOPATIA. |
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| ES200401428A Expired - Fee Related ES2317713B1 (en) | 2004-06-07 | 2004-06-07 | METHOD FOR DIAGNOSING OR DETERMINING THE GENETIC PREDISPOSITION TO DEVELOP HYPERTROPHIC MIOCARDIOPATIA. |
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| CN102242212B (en) * | 2011-07-08 | 2013-04-10 | 泰普生物科学（中国）有限公司 | HCM (Hypertrophic Cardiomyopathy) genotyping method and kit |
| CN108642155A (en) * | 2018-06-15 | 2018-10-12 | 合肥艾迪康临床检验所有限公司 | Detect method, oligonucleotides and its application of MYH7 gene mutations |
| CN112941175B (en) * | 2021-04-14 | 2022-06-17 | 大理大学 | Primer probe composition for detecting MYH7 gene mutation and application thereof |
| CN112941172B (en) * | 2021-04-14 | 2022-05-31 | 大理大学 | Primer probe composition for detecting TNNC1 gene mutation and application thereof |
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| US20020127548A1 (en) * | 1992-12-11 | 2002-09-12 | Christine Siedman | Methods for detecting mutations associated with hypertrophic cardiomyopathy |
| US5912121A (en) * | 1992-12-11 | 1999-06-15 | Bringham And Women's Hospital | Methods for detecting mutations associated with hypertrophic cardiomyopathy |
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Non-Patent Citations (3)
| Title |
|---|
| ENJUTO M. et al. "{}Malignant hypertrophic cardiomyopathy caused by the Arg723Gly mutation in beta-myosin heavy chain gene"{}. Journal of molecular and cellular cardiology. Dic. 2000. Vol. 32, N$^{o}$. 12, páginas 2307-2313. ISSN 0022-2828 (Print). * |
| MOOLMAN-SMOOK JOHANNA C. et al. "{}The origins of hypertrophic cardiomyopathy-causing mutations in two South African subpopulations: A unique profile of both independent and founder events"{}. American Journal of Human Genetics. Nov. 1999. Vol. 65, N$^{o}$. 5, páginas 1308-1320. ISSN 0002-9297. * |
| MÖRNER STELLAN et al. "{}Identification of the genotypes causing hypertrophic cardiomyopathy in northern Sweden"{}. Journal of molecular and cellular cardiology. Jul. 2003. Vol. 35, N$^{o}$. 7, páginas 841-849. ISSN 0022-2828 (Print). * |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2339841B1 (en) | 2011-02-28 |
| ES2340459A1 (en) | 2010-06-02 |
| ES2317713A1 (en) | 2009-04-16 |
| ES2339841A1 (en) | 2010-05-25 |
| ES2317713B1 (en) | 2009-12-01 |
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