WO2003074737A1 - Nouveau procede - Google Patents

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Publication number
WO2003074737A1
WO2003074737A1 PCT/SE2003/000394 SE0300394W WO03074737A1 WO 2003074737 A1 WO2003074737 A1 WO 2003074737A1 SE 0300394 W SE0300394 W SE 0300394W WO 03074737 A1 WO03074737 A1 WO 03074737A1
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primer
extension
nucleic acid
primers
acid molecule
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PCT/SE2003/000394
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English (en)
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Anna-Lotta Schiller
Jenny Dunker
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Biotage
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Priority to AU2003210099A priority Critical patent/AU2003210099A1/en
Publication of WO2003074737A1 publication Critical patent/WO2003074737A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention refers to a method for typing of genetic material related to cystic fibrosis using primer extension, a method for diagnosis of the genetic predisposition of cystic fibrosis and states associated therewith, as well as a kit for performing the methods.
  • Cystic fibrosis an autosomal recessive disorder, is the most prevalent lethal genetic disease of Northern European populations, affecting 1 live birth in 1600 to 2500.
  • the carrier rate is therefore quite high, about 1 out of every 25 Caucasians.
  • the disorder is less common in Southern Europe, and very much less frequent among native African and Asian populations, where it occurs in fewer than 1 in 100,000 births.
  • the European gene has spread, by the normal processes, into populations, which prefer to regard themselves as African- American (carrier rate: 1 out of 69) or Ashkenazi Jewish (1 out of 29).
  • Cystic fibrosis is essentially a disease of the epithelial cells. It is characterised by abnormal exocrine gland excretion, insufficiency of the pancreas (where the fibrous cysts that give the name occur), increased sweat salt content, over-production of extremely sticky bronchial mucus and consequent chronic lung disease. About 85 % of the male carriers are sterile, and female fertility is much reduced. Patients with a severe form of the disease are likely to die as children (the average life span for a carrier being about 30 years), usually of lung disorders; milder cases may have no worse problems than sterility. However, sterility is not a mandatory effect of the disease.
  • cystic fibrosis transmembrane conductance regulator CFTR
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the gene is expressed in epithelial cells, though often the level of transcription of the gene is low; in bronchial epithelial cells, only one copy of mRNA per cell is present, though there are more in the submucosal glands which produce the mucus that is so superabundant in CF patients.
  • a person In order to get the disease a person must be carrier of at least two mutations, one on each chromosome (one from the father and one from the mother). To develop the disease a carrier may be homozygote (the same mutation on both chromosomes) or "compound heterozygote" (two different mutations on different chromosomes, which together give rise to CF (for instance two heterozygotes deltaF508 and G85E)). The most common mutation is delta F508, accounting for approximately 70% of all mutations.
  • SSCP single strand conformation polymorphism
  • direct sequencing of the mutant DNAs have been used to screen patient samples.
  • chemical cleavage mismatch technique has been used to screen for mutations and to identify known mutations in samples (Jones et al., Human Molecular Genetics, 1(1): 11-17).
  • a multiplexed assay to detect wild-type and mutant DNA sequences in the CFTR gene using the LabMAP system has been developed (Dunbar and Jacobson, Clinical Chemistry, 2000; 46:1498-1500), wherein a plurality of microspheres having varying specific detectable spectral addresses can be analysed in a single reaction vessel.
  • CFTR gene mutations have been detected using primer oligo base extension and mass spectroscopy (Braun et al., Clinical Chemistry, 1997; 43:1151-1158).
  • a single mutation detection primer is annealed to the target nucleic acid downstream of a mutation region, and the primer is allowed to extend over the mutation region.
  • the product lengths are determined from their molecular mass in MS, thereby allowing determination of the place and nature of the mutation(s).
  • Ronaghi et al. discloses the use of one primer for the detection of two single nulceotide polymorphisms that are positioned about 50 nucleotides apart from each other on the same nucleic acid molecule.
  • DNA may be sequenced with the pyrosequencing method.
  • the method may be used for typing e.g. cystic fibrosis.
  • the prior art technology does therefore not disclose how to solve the problem of determining, in a reliable and reproducible way, a plurality of single nucleotide polymorphisms in a genetic material, such as the gene coding for cystic fibrosis.
  • more than one variable site is analysed in the same reaction, by providing more than one extension primer, as well as one or more PCR-templates, wherein the different primers are adapted for the detection of different variable sites.
  • the occurrence of several mutations may be screened for in the same reaction vessel, allowing a fast analysis and detection of multiple mutations.
  • the number of interpretable nucleotides in one reaction is increased.
  • the number of nucleotides that can be analysed are about 2 x 70, i.e. about 140 nucleotides, and so on for each new primer that is used.
  • What limits the analysis when using more than one primer is actually the composition and sequential order of nucleotides on the nucleic acid molecule, and hence the chosen dispensation order and extension primer design. Accordingly, when using more than one primer in each reaction vessel, the total number of reactions for analysing a large number of mutations in the CFTR-gene is greatly reduced compared to the technologies of the prior art.
  • extension primer if more than one extension primer is designed to anneal to the nucleic acid molecule that is to be analysed in such a way that the distance between the annealed primers are in the order of 50-70 nucleotides (i.e. the number of nucleotides that is possible to read with one primer), then a corresponding distance of the nucleic acid molecule would be typed.
  • four extension primers are designed and used in this way, it would be possible to analyse a stretch of the nucleic acid molecule corresponding to about 200-250 nucleotides. The same reasoning would apply for another number of primers.
  • the invention refers to a kit comprising extension primers, which are specifically designed for analysis of known mutations in the CFTR- gene by the method described above.
  • Figure 1-11 refers to assay design for detection of variable sites in exon 10 and 11 according to the method of the invention.
  • Figure 12 and 13 show the theoretical output from PyrosequencingTM reactions.
  • typing is meant any method for analysing the nucleotide sequence of the nucleic acid molecule to be analysed. Specifically, typing methods of the invention include methods for detecting, identifying or analysing genetic or sequence variation in one or more target nucleic acid(s). By “genotyping” is meant to determine the genotype of the target nucleic acid molecule. The genotype, in turn, refers to a combination or pattern of multiple genetic variations (or “variable sites") in the target nucleic acid.
  • a nucleic acid molecule is meant any suitable stretch of DNA, originating from the CFTR gene, which is long enough to allow detection of at least two variable sites.
  • the nucleic acid molecule may be isolated or synthesised, in any convenient form. Thus, it may be genomic DNA, isolated mRNA, cDNA, a PCR-product or in any other possible form, such as being obtained from a clone, or being a library product.
  • the nucleic acid molecule(s) may be obtained or derived from any convenient source, which may be any material containing nucleic acid of the CFTR gene. For instance, this may be a clinical sample, a cell line, a tissue sample, an amniocentesis, or a blood sample from an individual.
  • the nucleic acid may be provided for investigation in any convenient form and conveniently will be contained in a sample, e.g. an aqueous sample.
  • the nucleic acid may be prepared for the typing method, as desired, according to techniques well known in the art, e.g. isolation, purification, cloning, copying, amplification, etc.
  • the invention relates to a method for typing of at least one nucleic acid molecule of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene comprising the steps of:
  • nucleic acid molecule of the CFTR gene provides at least one nucleic acid molecule of the CFTR gene; (ii) providing at least one extension primer, which primer(s) bind(s) to a (different) predetermined sites in the nucleic acid molecule(s), wherein at least one extension primer is designed to extend over at least two potential variable sites in the nucleic acid molecule(s), and nucleotides; (iii) simultaneously or sequentially performing primer extension reactions, and determining the pattern of nucleotide incorporation to obtain a test pattern;
  • step (iv) optionally comparing the test pattern of step (c) with one or more reference pattern(s), in order to type the variable sites of the nucleic acid molecule(s).
  • variable site is typed by the use of a single extension primer.
  • extension primers primers that are able to anneal to the nucleic acid molecule, and which allow incorporation of nucleotides at their 3 '-end.
  • An extension primer bind to the target nucleic acid at a predetermined site, each primer binding site being different, so that multiple different primer extension reactions are performed.
  • the extension primers are designed or selected so that their extension products overlap (or comprise) a site (e.g. a locus or region) of sequence variability (i.e. genetic variation) in the target nucleic acid. In other words, the primers bind to the target nucleic acid at, or near to (e.g.
  • the extension primers are chosen from the group comprising extension primers which are designed for at least two of the variable sites chosen from the column of table 1 (figure 12) that is referred to as "Mutations screened in kit B", whereby the extension primers have a length of 5 to 40 nucleotides, preferably 10-20, and will be designed to be complementary to a sequence of the CFTR gene within an interval of 1-20 nucleotides, preferably 1-5, upstream or downstream from the chosen variable site(s).
  • At least one extension primer is designed to be able to extend over at least two variable sites, thereby allowing the typing of at least two variable sites with only one extension primer.
  • At least one extension primer is required to carry out the method of the invention, and in one embodiment at least two.
  • the number of primers may be varied according to choice, for example, depending on the complexity of the system under study, and the detail of the information it is desired to obtain.
  • 3, 4, 5, or 6, or more extension primers e.g. 3 to 15, or 3-10) may be used.
  • more than one extension primer is used in the same reaction. This will make it possible to increase the number of interpretable nucleotides in a single reaction. For instance, if two extension primers are used in the same reaction, twice as many nucleotides may be analysed as when using one primer. When using more than one primer it is important to choose the dispensation order and the primer design in such a way that the data from the reaction is interpretable. How this is done can be seen in the Example section.
  • the method of the invention for genetic analysis of at least two sites simultaneously works in the following way: Firstly, with one primer, the sequence will be deduced from the detection of released pyrophosphate after incorporation of a nucleotide triphosphate. The height of the light signal is proportional to the number of incorporated nucleotides (a profile with peaks is generated (a pyrogram)). Secondly, with two primers present in the same reaction mix, two such profiles (or pyrograms) will be superimposed on top of each other, i.e. if the bases to be sequenced after primer 1 is ATG...
  • primer 2 when the A is incorporated a peak with the relative height of 1 will be generated, and if the next added base is a C, primer 2 will give a signal with the relative height of 1, but when the T is added the signal will be twice the relative height (since both primers will be extended simultaneously and thus give rise to a signal). Finally, the same will occur when there are three or more primers participating in the same reaction. Different combinations of samples will thus give different profiles, and by comparing them with different theoretical profiles, the genotype or the sequence for the stretches of the nucleic acid over which the extension primers are extended can be deduced simultaneously, and thereby both reagents and time can be saved when analysing a large number of samples or longer parts of DNA.
  • the length of the nucleic acid molecule(s) is in the interval from 50-3000 nucleotides.
  • nucleotides are meant any conventional nucleotides that are used in reactions and methods of this kind, that is any normal or modified variants of the nucleotides corresponding to the bases A, G, C, T and U. More detail is given below.
  • reagents are optionally included in the primer extension reaction, such as those reagents, which are normally used for protocols of this kind. Normally, for instance, a polymerase enzyme is included.
  • suitable and necessary reagents will be obvious from the Example section of this text.
  • PCT/GB01/64042 describes various modifications and explanations concerning the methods and the reagents used in this invention. This document is hereby incorporated as reference.
  • determining the pattern of nucleotide incorporation is meant to be able to monitor or detect in what way nucleotides are incorporated and how the extension primers are extended towards the 3 '-end. This may be achieved by any suitable method. Preferably, sequencing-by-synthesis is used (see e.g. US-A-4863849) to determine the pattern of nucleotide incorporation.
  • Sequence-by-synthesis refers to sequencing methods, which rely on the detection of nucleotide incorporation during a primer-directed polymerase extension reaction.
  • the four different nucleotides i.e. A, G, T or C nucleotides
  • A, G, T or C nucleotides are added cyclically or sequentially (conveniently in a known order), and the event of incorporation can be detected in various ways, directly or indirectly. This detection reveals which nucleotide has been incorporated, and hence sequencing information.
  • base which forms a pair (according to the normal rules of base-pairing, A-T and C-G) with the next base in the template target sequence, is added, it will be incorporated into the growing complementary strand (i.e. the extended primer) by the polymerase, and this incorporation will trigger a detectable signal, the nature of which is dependent upon the detection strategy selected.
  • the primer extension reactions occur simultaneously, i.e. both or all primers are annealed and are capable of primer extension at the same time.
  • each individual primer can only be extended if a nucleotide is added to the reaction mix, which is complementary to the next nucleotide in the template.
  • the term "simultaneous" must be interpreted with this in mind.
  • test pattern (or sequence pattern) is meant the pattern of nucleotide incorporation that is achieved from the nucleic acid molecule(s) that are subjected to the primer extension reactions.
  • reference pattern is meant a pattern achieved from another primer extension reaction, or a theoretically produced pattern, which pattern may be compared to the test pattern, and thereby, for instance, making it possible to distinguish the identity of variable sites.
  • variable sites are meant positions (e.g. loci or regions) in the nucleic acid molecule, which, in comparison to another variant of the nucleic acid molecule, such as an allele or the like, show variety. This means that some kind of polymorphism, insertion, deletion, addition or substitution is present in at least one of the compared nucleic acid molecules, thereby making the nucleic acid molecule(s) different from one another at these sites.
  • a variable site can differ in different genotypes.
  • the variable sites under focus here are mainly disclosed in table 1. Accordingly, each "type” will comprise a region of sequence variation, wherein this region (i.e. the sequence, or base identity, at that site) can be different from other types.
  • At least two potential variable sites are examined, and, when one target nucleic acid molecule is typed, said nucleic acid molecule thus contains 2 or more (i.e. multiple) variable sites. Where 2 or more target nucleic acid molecules are typed, said nucleic acid molecules thus each contain 1 or more variable sites.
  • variable sites can be analysed by the method of the invention, as long as they are comprised within the CFTR gene.
  • the variable sites may be within a coding region or a non-coding region of the CFTR gene.
  • the variable site can be of any length.
  • the variable site may comprise only a single or a few (e.g. 1-6, e.g. 1, 2, 3, 4, 5 or 6) nucleotides at which the sequence of the target nucleic acid may be variable.
  • primer binding sites should be available in all possible variants (genotypes) of the nucleic acid molecule(s) under study. Such primer binding sites will therefore advantageously lie in regions, which are common to, or substantially conserved between, the different variants. This may readily be achieved by selecting the primer binding sites to lie in conserved/ semi- conserved regions.
  • the primer extension reactions conveniently may be performed by sequentially adding the nucleotides to the reaction mixture (i.e. a polymerase, and primer/template mixture).
  • the different nucleotides are added in known order, and preferably in a pre-determined order. As each nucleotide is added, it may be determined whether or not nucleotide incorporation takes place.
  • the amount of each nucleotide incorporated is determined, since the output signal is proportional to the incorporated number of nucleotides, i.e. two incorporated nucleotides with the same base will give twice as high signal as one incorporated nucleotide etc.
  • the pattern of nucleotide incorporation may be determined.
  • the step of determining the pattern of nucleotide incorporation may comprise determining (or detecting) whether or not, and which, nucleotide is incorporated.
  • a "pattern” may be obtained for the target nucleic acid.
  • This pattern comprises the base identity (i.e. sequence) of the particular variable sites identified for that nucleic acid molecule.
  • the pattern corresponds to the genotype of the target nucleic acid.
  • the genotype may readily be identified by comparing the obtained pattern to a reference pattern (or a "standard pattern"), or a panel of reference patterns (i.e. one or more, e.g. two or more, e.g. 1 to 100, 1 to 20, 1 to 15, 1 to 10, 1 to 6 or 1 to 3).
  • a reference pattern may readily be obtained by determining the pattern of nucleotide incorporation using the extension primers in question on reference nucleic acid molecules of known genotype (e.g. a known polymorphic pattern).
  • the "reference pattern" can be theoretically derived from knowledge of the variable sites. Actually, it may then not be necessary to compare the pattern obtained with a reference pattern, the desired typing/sequence information can be read from the pattern obtained. Once the extension primers for each variable site have been selected and the order of addition of nucleotides determined, it is possible to determine a theoretical output from a primer extension reaction.
  • the genotype of the molecule may be identified (or recognised).
  • test patterns and reference patterns may be compared using pattern recognition software.
  • amplification primers are designed and used, which primers are designed to be suitable for producing the desired nucleic acid molecule.
  • the amplification primer(s) may be used as extension primer(s).
  • sequence and length of the amplification and extension primers will depend on the sequence of the target nucleic acid, the desired length of the product, the possible further functions of the primer (i.e. for immobilisation) and the method used for amplification and/or extension.
  • Advantageously extension primers will bind near (e.g. within 1-40, 1-20, 1-10 or 1-6, preferably within 1-3 bases), substantially adjacent to the variable site of the target nucleic acid and will be complementary to a conserved or semi-conserved region of the nucleic acid.
  • an extension primer is provided for each of the variable regions, each being specific for a site at or near to the variable site. The specificity is achieved by virtue of complementary base pairing.
  • primer design may be based upon principles well known in the art. It is not necessary for the extension or amplification primer to have absolute complementarity to the binding site, but this is preferred to improve the specificity of binding. Further, the extension primer may be designed to bind to the sense or anti-sense strand of the target nucleic acid.
  • extension primers are designed in such a way as to allow the typing of each variable site to occur discretely. This means that analysis of a given variable site not is disturbed by the positive signal from the nucleotide incorporation at another site.
  • the "primer extension” reaction includes all forms of template-directed polymerase-catalysed nucleic acid synthesis reactions. Conditions and reagents for primer extension reactions are well known in the art, and any of the standard methods, reagents and enzymes etc. may be used in this step (see e.g. Sambrook et al., (eds), Molecular Cloning: a laboratory manual (1989), Cold Spring Harbor Laboratory Press). Thus, the primer extension reaction at its most basic is carried out in the presence of primer, deoxynucleotides (dNTPs) and a suitable polymerase enzyme e.g. T7 polymerase, Klenow or Sequenase Ver 2.0 (USB USA), or indeed any suitable available polymerase enzyme. For an RNA template, reverse transcriptase may be used. Conditions may be chosen with regard to well-known procedures in the art.
  • the primer is subjected to a primer extension reaction in the presence of a nucleotide, whereby the nucleotide is only incorporated if it is complementary to the base immediately adjacent (3') to the primer position.
  • the nucleotide may be any nucleotide capable of incorporation by a polymerase enzyme into a nucleic acid chain or molecule.
  • the nucleotide may be a deoxynucleotide (dNTP, deoxynucleoside triphosphate) or dideoxynucleotide (ddNTP, dideoxynucleoside triphosphate).
  • nucleotides may be used in the primer extension reaction: guanine (G), cytosine (C), thymine (T) or adenine (A) deoxy- or dideoxynucleotides. Therefore, the nucleotide may be dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate), dTTP (deoxythymidine triphosphate) or dATP (deoxyadenosine triphopshate). Suitable analogues of these nucleotides may also be dye-labelled n.t, such as dye-dNTP, dye-ddNTP or the recently described dye-SS-dNTP (WO00/53812).
  • Dideoxynucleotides may also be used in the primer extension reaction.
  • the term "dideoxynucleotide” as used herein includes all 2 '-deoxynucleotides in which the 3'- hydroxyl group is modified or absent. Dideoxynucleotides are capable of incorporation into the primer in the presence of the polymerase, but cannot enter into a subsequent polymerisation reaction, and thus function as a chain terminator.
  • nucleotide incoiporation takes place at one variable site, no nucleotide incorporation takes place at the other variable site(s).
  • nucleotide incorporation is determined quantitatively. This is advantageous when several nucleotides of the same kind are incorporated directly after each other, thereby making it possible to determine the number of incorporated nucleotides of the same kind.
  • Another way of making it possible to type more than one variable site in one reaction using more than one extension primer is to choose the extension primers in a way that the nucleotide incorporation at different variable sites not occur simultaneously. Accordingly, in one embodiment a first extension primer binds closer to its variable site, than a second extension primer does to its variable site. In a still more preferred embodiment, the second primer is 2-10 nucleotides further away from its variable site, than the first primer is from its variable site.
  • reaction conditions for using more than one extension primer compared to using one extension primer in a reaction vessel are basically the same.
  • the two extension primers can be located at the same distance from the variable site.
  • the variable site is solved by the order of added nucleotides, i.e. the dispensation order, preferably in a sequencing-by-synthesis system.
  • the invention allows for the typing of even more than two variable sites in one reaction using more than one extension primer. Accordingly, in one embodiment at least 3 variable sites are typed. Hereby, at least 3 primer extension reactions may also be performed.
  • PCT/GB 01/64042 of Pyrosequencing AB is incorporated as reference.
  • the example section discloses one advantageous technique for performing the typing and the detection of the nucleotide incorporation, which technique is referred to as PyrosequencingTM, which is described below.
  • Pyrosequencing is a sequencing method developed at the Royal Institute of Technology in Sweden (Ronaghi et al.,1998, Alderborn et al.,2000). The method is based on "sequencing by synthesis" in which, in contrast to conventional Sanger sequencing, the nucleotides are added one by one during the sequencing reaction.
  • An automated sequencer, the PSQ96TM instrument has recently been launched by Pyrosequencing AB (Uppsala, Sweden).
  • the principle of the pyrosequencing reaction A single stranded DNA fragment (attached to a solid support), carrying an annealed sequencing primer acts as a template for the pyrosequencing reaction. In the first two dispensations, substrate and enzyme mixes are added to the template.
  • the enzyme mix consists of four different enzymes; DNA polymerase, ATP-Sulfurylase, Luciferase and Apyrase.
  • the nucleotides are sequentially added one by one according to a specified order dependent on the template and determined by the user. If the added nucleotide is matching the template, the DNA polymerase will incorporate it into the growing DNA strand. By this action, pyrophosphate, PP i5 will be released.
  • the ATP-Sulfurylase converts the PPi into ATP
  • Luciferase transforms the ATP into a light signal.
  • the fourth enzyme Apyrase
  • the template will at that point be ready for the next reaction cycle, i.e. another nucleotide addition. Since no PPi is released unless a nucleotide is incorporated, a light signal will be produced only when the correct nucleotide is incorporated.
  • the PSQ 96 Instrument has been developed by Pyrosequencing AB (Uppsala, Sweden) in order to automate the sequencing reaction and to monitor the light release.
  • the PSQ 96 Instrument software presents the results as peaks in a pyrogramTM, where the height of the peaks corresponds to the number of nucleotides incorporated.
  • PyrosequencingTM is a real time DNA sequencing method based on sequencing-by- synthesis. The method is proved to be a fast and accurate method for SNP (single nucleotide polymorphism) scoring, sequencing of shorter DNA stretches (signature tags), and assessment of allele frequencies.
  • Pyrosequencing AB manufactures the PSQTM 96 and the PTP Systems for low and high throughput genotyping, respectively, as well as dedicated softwares for automatic delivery of genotype and a quality assessment for each sample. A major advantage with those Systems is the combination of accuracy, speed and ease-of-use.
  • the method of the invention is conveniently performed in a single reaction vessel, whether a sequential or simultaneous primer extension is performed.
  • all extension primers may be added simultaneously or sequentially into a single reaction vessel.
  • a sequential primer extension protocol When a sequential primer extension protocol is used, a first extension primer is added, whereby the primer extension is performed until its variable site(s) has been incorporated with a nucleotide. Then a chain terminator is added, such as a dideoxynucleotide. Thereafter, the second extension primer is added and the second primer extension is performed. Additionally, primer extensions may of course also be performed.
  • a simultaneous primer extension protocol all extension primers are added together. In this case the primer design and the dispensation order of the nucleotides is preferably chosen in such a way, that nucleotide incorporation to different variable sites does not occur at the same time.
  • the nucleic acid molecule for use in the method is provided in a single stranded format. This is achieved by standard techniques. However, it may also be provided in a double stranded format into the reaction vessel, and thereafter subjected to strand separation by any suitable technique in the art (see e.g. Sambrook et al.)
  • the target nucleic acid may be provided with a means for immobilisation, which may be introduced during amplification. Techniques to perform this are well known in the art, and for a more detailed disclosure US-A-5405746, US-A-6210891 and PCT/GB 01/64042 are incorporated as reference.
  • the inventive method is for example advantageous in that it determines the exact sequence of the variable sites (i.e. is based on sequencing procedure), it avoids costly and cumbersome procedures, such as electrophoresis, and advantageously labelled nucleotides and or primers, and large numbers of samples can be analysed in short time.
  • CCC homopolymeric stretches
  • This may be overcome by the addition of a single-stranded nucleic acid binding protein (SSB) once the extension primers have been annealed to the template nucleic acid.
  • SSB single-stranded nucleic acid binding protein
  • the use of SSB in sequencing-by-synthesis is disclosed in WO00/43540 of Pyrosequencing AB.
  • the invention refers to a method for diagnosis of various states and diseases, which are coupled to a genetic predisposition in the CFTR gene. These states are at foremost various symptoms and states related to cystic fibrosis, as referred to above, and may for example include diseases coupled to breathing and digestion.
  • the method for diagnosis according to the invention comprises the steps of:
  • step (b) providing at least one nucleic acid molecule of the human CFTR gene from the material in step (a); (c) typing the nucleic acid molecule(s) of step (b) according to the method of the invention, in order to determine the genetic predisposition for CF for the chosen individual.
  • the invention refers to a kit for use in the method of the invention, comprising at least one extension primer, which is designed for different variable sites.
  • the kit optionally comprises at least two primers for in vitro amplification, at least one nucleic acid molecule chosen from the CF gene, at least one extension primer, which bind to the nucleic acid molecule(s), nucleotides for amplification and/or for primer extension reactions, optionally a polymerase enzyme for amplification and/or for primer extension reactions, as well as other suitable and necessary reagents, and optionally means for detecting the primer extension.
  • the kit of the invention comprises a set of primers, at least two, allowing the detection of a panel of mutations related to cystic fibrosis.
  • the set of primers includes the primers, which are designed for analysing the variable sites referred to in table 1 as "Mutations screened in kit B".
  • the set of primers includes the primers, which are designed for analysing the variable sites referred to in table 1 as "Mutations screened in kit A”.
  • the set of primers include the primers of column G and H of table a ("Sequencing primers").
  • a kit is provided, allowing the detection of a panel of mutations related to cystic fibrosis.
  • any known mutation related to cystic fibrosis may be detected in this way, as long as an extension primer designed for the detection of the specific mutation is included in the kit.
  • any new mutations in the CFTR-gene not known today, may be detected by using a kit of the invention. This allows for rapid typing of genes related to the CFTR gene, and thereby the possible diagnosis of diseases related to the CFTR gene and/or drug dose optimisation for different individuals.
  • Table 1 discloses the variable sites to be covered by the extension primers of kit A and kit B, respectively, and the sequences of the proposed extension primers for respective variable site.
  • means for detecting the primer extension are meant any means, which allows for the detection and analysis of the primer extension. This may in a preferred embodiment involve the sequencing-by-synthesis system, but other means and systems may also be used.
  • some of the main features of the invention are that it allows for a design and choice of extension primers, as well as a predetermined dispensation order of nucleotides in the primer extension reactions, making it possible to analyse and type more than one variable site in one or more nucleic acid molecule(s) in a single reaction vessel, optionally by using one or more extension primers in each reaction vessel.
  • Example section shows the generation of extension primers for various variable sites in the CFTR gene, as well as the detection of multiple variable sites using the method of the invention.
  • I506V I507V 1 & F508O 5T/7T/9T
  • the 5T variant can also cause CF in combination with Rl 17H.
  • the Committee recommends that since a patient with a negative result still has a residual risk for CF heterozygosity depending on her/his ethnic or racial background, the provider will be responsible for informing the screenee and couple of their residual risk. Such calculations are estimates based on the patient's and partner's ethnic and racial background. This information can be provided for each screenee in the test report.
  • certain mutations are associated with infertility and may require additional testing for appropriate interpretation and genetic counseling. Evidence has been presented that children with CF identified by newborn screening have earlier nutritional advantages compared with those in a non-screened group. Data also indicate a slower deterioration of lung function in screened patients (Schaedel et al, 1999).
  • PCR amplification A 50 ⁇ l PCR reaction was set up for each exon-specif ⁇ c fragment and sample. All fragments were amplified with the Qiagen PCR amplification kit (Qiagen) using either 1.5 or 3.0 mM MgCl 2 (see Table 3) according to the following protocols.
  • Sample preparation was performed according to the protocol recommended by Pyrosequencing. Immobilization of the biotinylated PCR product to Dynabeads M-280 Streptavidin (Dynal AS) was performed at 65°C, 1400 RPM for 15 minutes. The immobilization mixture for all SNPs consisted of: 20 ⁇ l PCR product + 10 ⁇ l Dynabeads (10 ⁇ g/ ⁇ l) + 25 ⁇ l 2xBW buffer. Annealing of sequencing primer to the templates was performed with 15 pmol sequencing primer, for 2 minutes at 80°C. The samples were allowed to cool to room temperature before sequencing in PSQ96TM system.
  • Table 6 summarises the various assays that were made with one extension (or sequencing) primer per reaction. In the table it can be seen how many variable sites that were analysed by each extension primer for the various exons and introns of CFTR that were analysed.
  • the primer should fulfil the criteria that the 3 '-end is positioned 1-40 bases upstream or downstream of the mutation to be studied, whereby the orientation depends on which strand is analyzed. More preferably, the 3 '-end of the primer should be within 1-3, or 1-5, or 1-10 or 1-30 bases upstream or downstream of the mutation.
  • the selection of primers is also dependent on the dispensation order of the different nucleotides.
  • Table 7 summarises the assays that were made with more than one extension primer per reaction.
  • the reaction conditions are as defined above for simplex assays.
  • PCR assays there will be possible to have two different approaches concerning the PCR assays: (1) Separate PCR amplifications of each exon. This will result in 15 PCR products, 15 extra negative controls in the pyrosequencing assay and in prolongation of this 15 addition vials in the kit. (2) Multiplex PCR amplifications of either one 15-plex PCR or two 7-8 multiplex PCRs. This will result in only 1-2 extra vials in the kit as well as a reduced number of negative controls in the assay.
  • pancreatic insufficiency >95% of all cases
  • young age diagnosis usally ⁇ 1 year of age
  • high sweat chloride levels >80 meq/1
  • meconium ileus (20-30% of all cases).
  • PS pancreatic sufficiency
  • I507V and I508V have not been associated with CF; however, F508C has been reported to be associated with CBAVD. Although the frequency of these variant alleles is rare, occasionally an otherwise healthy CF carrier, who also has the rare variant, may be erroneously diagnosed as an affected homozygote.
  • Figure 1- 11 refers to assay design for exon 10 (and exon 11) and mutation screening of the mutations delta508F and delta507F, as well as some other mutations (indicated in the figure).
  • the principle for primer design for analysing more than one mutation with a single primer is demonstrated.
  • Columns A and B show the core mutation panel, i.e. the set of 25 mutations that are frequently analysed in typing of Cystic fibrosis.
  • Columns C and D show the mutations that are screened in kit A of the invention (47 mutations).
  • Columns E and F shows the mutations that are screened in kit B of the invention (89 mutations).
  • the respective sequencing primer(s) for each mutation that are screened in kit A and B are shown in columns G and H. For some mutations more than one suitable primer is shown.
  • reflex test data are also shown, at the bottom of the table.
  • PyrosequencingTM has proven applicable for identifying previously mapped SNPs in a quick and easy way.
  • the strength of this technology is now further enhanced by multiplex genotyping, where two or more SNP positions are being analyzed in the same reaction.
  • the most obvious advantages with this new course of action are the savings of reagents and the cutting-down of analysis time.
  • the goal of multiplex Pyrosequencing is to genotype several SNP positions in a single reaction. This is accomplished by sequencing several fragments in a single well using a unique sequencing primer for each desired SNP.
  • the resulting pyrogramTM pattern will be a mix of the different sequences overlapping each other ( Figure 12).
  • the assays are designed to generate unique peaks for each fragment in the multiplex genotyping reaction. It is important that one or two polymorphic peaks as well as at least one single peak, which will serve as a reference, from each fragment can be analyzed without simultaneous nucleotide incorporation from the other fragment(s).
  • the heights of the polymorphic peaks are related to the reference peak for the same fragment to secure highest possible accuracy.
  • Multiplex Pyrosequencing can be carried out at different levels. It is most time- and cost- efficient if multiplex Pyrosequencing is preceded by a multiplex PCR. It is also possible to pool simplex PCR products and multiplex only the Pyrosequencing reaction. Finally, a third option is to amplify one fragment including several polymorphic sites and then design several sequencing primers, which are all used for sequencing in the same reaction.
  • the sequencing primers are designed simultaneously with the dispensation order.
  • One example that illustrates how to proceed step by step is given below. Note that each case is individual and can be solved in many ways!
  • the following dispensations should be selected such that SNP 2 is analyzed and at least one reference peak for fragment 2 is obtained. It is important to try to avoid "overlapping of peaks" in the SNP position, i.e. that peaks originating from fragment 1 interfere with the peaks arising from fragment 2, in the SNP 2 dispensations.
  • a multiplex Pyrosequencing ran is carried out in the same way as a normal simplex run with respect to sample preparation. The final total amount of sample needed is the same. It should be noted that if simplex PCR samples are pooled prior to multiplex Pyrosequencing, an optimization of volume needed from each sample might be necessary if peaks of the same height from both fragments are desired. This is, however, not necessary.
  • sequencing primer design is limited by the fact that the 3 ' end positions are fixed. This can make it difficult to avoid the formation of sequencing primer dimers with 5' overhangs just by re-designing the sequencing primers.
  • a trick to come around this problem is to include an additional step in the sample preparation. This step is included after annealing at 80 °C for 2 minutes. Instead of placing the plate into the instrument, magnetic beads are caught with the PSQTM 96 Sample Prep Tool once again and moved to 40 ⁇ l of fresh IX Annealing buffer. If Sepharose beads are used, the samples are moved back to a filter plate after primer annealing. Apply vacuum to remove the liquid with excess primer dimers and then wash twice before resuspending in fresh annealing buffer. Finally transfer beads to a PSQ 96 Low plate. Thereby dimers causing background will be left behind and will not interfere with the Pyrosequencing reaction.
  • Single-stranded DNA-binding protein is a protein, which can displace weak interactions between single-stranded DNA molecules and loosen up secondary structures.
  • SSB Single-stranded DNA-binding protein
  • Some examples are background caused by mispriming of a sequencing primer to a template or the template looping back to itself.
  • SSB is added after annealing of the sequencing primers to the templates and then a run is started as normal. In order to deduce the amount of SSB needed for each individual assay, a titration is necessary. In general, 0.55 ⁇ g - 2.2 ⁇ g is sufficient.

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Abstract

L'invention concerne un procédé de typage d'au moins une molécule d'acides nucléiques du gène régulateur de la perméabilité transmembranaire de la fibrose kystique (CFTR) comprenant les étapes consistant: à produire au moins une molécule d'acides nucléiques du gène CFTR; à produire au moins une amorce d'extension conçue pour s'étendre sur au moins deux sites potentiels variables dans la/les molécules d'acides nucléiques; à exécuter de façon simultanée ou séquentielle des réactions d'extension d'amorce, et à déterminer le motif d'incorporation nucléotide en vue d'obtenir un motif d'essai; à éventuellement comparé le motif d'essai de l'étape (c) à un ou plusieurs motifs de référence, en vue de typer les sites variables de la/des molécules d'acides nucléiques. En outre, l'invention concerne un nécessaire comprenant un ensemble d'amorces utile dans les mutations de typage liées à la mucoviscidose. Cette invention fournit donc un système aisé, fiable et rapide permettant d'analyser un grand nombre de mutations liées à la mucoviscidose.
PCT/SE2003/000394 2002-03-07 2003-03-07 Nouveau procede WO2003074737A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054504A1 (fr) * 2003-12-02 2005-06-16 Universität Zu Köln Procede de sequençage multiplex
US20150211010A1 (en) * 2012-09-24 2015-07-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Restoration Of The CFTR Function By Splicing Modulation

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Publication number Priority date Publication date Assignee Title
WO1993018177A1 (fr) * 1992-03-13 1993-09-16 The Children's Hospital Of Philadelphia Diagnostic de la mucoviscidose par amplification enzymatique du genome multiplex specifique d'un allele
WO1998013523A1 (fr) * 1996-09-27 1998-04-02 Pyrosequencing Ab Procede de sequençage d'adn
EP0928832A2 (fr) * 1998-01-13 1999-07-14 Zeneca Limited Test pour la mucoviscidose basé sur la révélation de mutations dans le gène CFTR par ARMS

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WO1993018177A1 (fr) * 1992-03-13 1993-09-16 The Children's Hospital Of Philadelphia Diagnostic de la mucoviscidose par amplification enzymatique du genome multiplex specifique d'un allele
WO1998013523A1 (fr) * 1996-09-27 1998-04-02 Pyrosequencing Ab Procede de sequençage d'adn
DE69706430T2 (de) * 1996-09-27 2002-05-02 Pyrosequencing Ab Uppsala Verfahren zur sequenzierung von dna
EP0928832A2 (fr) * 1998-01-13 1999-07-14 Zeneca Limited Test pour la mucoviscidose basé sur la révélation de mutations dans le gène CFTR par ARMS

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Title
DATABASE WPI Week 200166, Derwent World Patents Index; Class B04, AN 1998-271708, XP002967391 *
RONAGHI MOSTAFA ET AL.: "Discovery of single nucleotide polymorphisms and mutations by pyrosequencing", COMPARATIVE AND FUNCTIONAL GENOMICS, vol. 3, 2002, pages 51 - 56, XP002967390 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054504A1 (fr) * 2003-12-02 2005-06-16 Universität Zu Köln Procede de sequençage multiplex
DE10356783A1 (de) * 2003-12-02 2005-07-07 Universität Zu Köln Verfahren zur Multiplex-Sequenzierung
US20150211010A1 (en) * 2012-09-24 2015-07-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Restoration Of The CFTR Function By Splicing Modulation
US10428328B2 (en) * 2012-09-24 2019-10-01 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Restoration of the CFTR function by splicing modulation
US10731156B2 (en) 2012-09-24 2020-08-04 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Restoration of the CFTR function by splicing modulation

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