CN101365944B - Methods for gene mapping and haplotyping - Google Patents
Methods for gene mapping and haplotyping Download PDFInfo
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Abstract
The present invention is directed to methods for providing a definitive haplotype of a subject. The haplotype information generated by the methods described herein is more accurate than that provided by prior art methods that only give an inferred haplotype. Accordingly, in one aspect the present invention provides a method for determining a definitive haplotype of a subject the method including the steps of providing a substantially isolated haploid element from the subject, and obtaining nucleotide sequence information from the haploid element. Applicants propose that the use of a substantially isolated haploid element eliminates the problem of incorrect or misleading inferences concerning the phase of two or more loci within a haplotype, and allows for revelation of two or more participatory genes within a haplotype, uncomplicated by differences in modes of inheritance. The guarantee of strictly cis-phase associations is provided in the present methods by the use of a substantially isolated haploid element as starting material for sequence analysis.
Description
The present invention relates generally to the genetics field.Tool more body is said the method that the present invention relates to gene mapping method and determination object haplotype.
Background of invention
Genome Atlas can be described order between gene on each karyomit(e) or other sign and the interval between them.Several different ratioss of human genome or the collection of illustrative plates of level of resolution have been made up.What resolving power was the most coarse is gene linkage map, is to describe their mode of inheritance by the relative position of each DNA sign (gene and other appraisable dna sequence dna) in karyomit(e).Gene linkage map has shown along the relative position of each specific DNA sign on the karyomit(e).The physiology of any heredity that can detect in the laboratory inequality or characterization of molecules namely are potential genetic markers between the individuality.This sign can be the DNA zone (gene) of expressing or do not have the known coded function but its mode of inheritance can be followed the DNA section.The difference of dna sequence dna is useful especially sign, because their feature rich and be easy to accurate evaluation.
Described sign must have polymorphism can be used for drawing collection of illustrative plates; That is to say, thereby multi-form must being present in all individualities can be detected them in the research between family's different members.Polymorphism is the variation in the dna sequence dna, and average every 300-500bp takes place once.Variation in the exon sequence can produce observable proterties and change, as the difference of eyeball color, blood group and disease susceptibility.Most of differences occur in the intron, and they have only minimum or not influence to the outward appearance of organ or function, but they can be detected thereby can be used as this sign at dna level.The sign example of these types comprises: the restriction fragment length polymorphism (RFLP) of sequence variations in the reflection DNA site of (1) available DNA restriction enzyme cutting, (2) difference of tandem repetitive sequence number, the tumor-necrosis factor glycoproteins of this weak point is because of the number difference length difference (a kind of feature that is easy to detect) of repeating unit.The Human genome linkage map be by observe two kinds of signs together heredity frequency how to make up.
Being positioned at two kinds of signs adjacent to each other on the same karyomit(e) tends to pass to child from mother together.Sperm and ovum are normal produce during, the dna double chain can rupture occasionally, on the same karyomit(e) or the different loci on another copy of same karyomit(e) (being homologous chromosomes) can connect.Two kinds of signs that this process (reduction division reorganization) can cause originally being positioned on the same karyomit(e) separate.The more closely linked possibility of sign close to each other is little, and the reorganization between them will be failed and be separated.Therefore recombination frequency can provide the estimation to distance between two kinds of signs.
The value of this kind gene mapping is can locate certain genetic diseases after certain DNA of diseased individuals existence indicates (but diseased individuals does not lack this sign) heredity in this figure, though may also not understand or not identify as yet the gene that it is responsible for disease to the molecular basis of this disease.Utilized genetic map to find to comprise the definite chromosomal localization of some important diseases genes of cystic fibrosis, sickle cell disease, Tay Sachs disease, fragile X mental retardation and myotonic dystrophy already.
The genetic method that influences gene function at present in the identified gene group has two common traits: at first, utilize the sign of non-coding sequence variation to point out the chromosomal region that contains candidate gene; Secondly, seek the sign of this kind sequence variations and the dependency between the phenotype interested by the genome extensive analysis.Finding gene by the extensive correlation analysis of genome, be also referred to as the linkage disequilibrium collection of illustrative plates, is U.S. Patent No. 5,851,762 theme.
Though utilized genomic information in the chain research, thought about the information of haplotype for the sign of identifying gene of interest group zone and the function in clear and definite (gene exists) disease is more useful.Employing haplotype sequence can reduce the mistake in the chain research, because do not need to consider that this gene relates to second of the proterties of studying the influence that (providing on the homologous chromosomes) causes is provided.
2003, the international association that NIH sets up has started one and has lasted 3 years projects of expensive 100,000,000 dollars and make human genome haplotype collection of illustrative plates (being called " HapMap "), to help by finding gene based on marker allele disease gene dependency in the complex disease of haplotype.This kind linkage disequilibrium allelotrope dependency collection of illustrative plates (' dependency collection of illustrative plates ') strategy relates to irrelevant individual patient, and for the traditional method of family's (pedigree) linkage analysis for can not find the whole family family time, this is a kind of more modern alternative method.
A kind of hypothesis about the HapMap basis is, thereby is enough to differentiate the assurance of dependency collection of illustrative plates as the continuity of the general gene discovery strategy of genome by the haplotype that diploid DNA gene type is inferred.
Another kind of hypothesis is, analysis to the SNP (single nucleotide polymorphism) of hundreds of individualities of four kinds of crowds (West Africa Black people, Japanese, Chinese, American) will be enough to identify redundant SNP, thereby be enough to identify the single SNP of haplotype mark, or minimize the tricks of SNP, thereby characterize everyone, comprise the haplotype parts among the mixing crowd.This HapMap aggregate (consortium) also point out in common multigenic disease and drug responsiveness in have only common haplotype (＞5-10%) be only most important, these common haplotypes will be in about 200 individual researchs in to identify.
Also having a kind of hypothesis is that monoploid has been formed different " parts (block) ", and each parts has an appraisable unique SNP " label ".What the genetics field was accepted at present is that the minimum of utilizing the HapMap scheme to disclose must be identified the enough common haplotypes of any crowd's mesopodium by SNP, with in the Disease-causing gene search be subjected to detect the excessive haplotype that has in the pharmacogenomics research of drug influence.
Therefore, the present state of this technology is that HapMap will be clear and definite, can provide information more fully for chain research.Yet, examine this HapMap scheme more nearly after, point out this scheme the best can only obtain finite information, may be invalid basically for the application that discloses multigenic disease.Perhaps even can not detect all common haplotypes for example, SNP identifies and can only detect the real haplotype of a part.And uncommon haplotype (HapMap may can not detect) also may be influential to the difference of gene function between the individuality.
More than Jia She monoploid block construction also may lead to errors.Estimate that reorganization and other rearrangement activity will influence the formation of haplotype parts among the mixing crowd, this be to " core " crowd's finite analysis can not disclose.
In a chromosomal region, there are two or more genes to have different modes of inheritance (recessiveness, dominance, codominance), have difference in functionality (susceptive disease, protection do not take a disease disease), when acting on the different steps of disease process, estimate that the resolving power of inferring the haplotype method may be challenged.When disease by complicated heterozygote latent disease for example due to two karyomit(e)s, and take place trans in the codominant inheritance gene (as those genes in the HLA complex body) and cis codominance when interacting, this resolving power will be subjected to ultimate challenge.The meaning of these uncertain queries is not also recognized in this area.
Identified the key gene of interest zone that to identify of detecting that utilizes haplotype dependency collection of illustrative plates by the pedigree linkage analysis.In an important detection example, the dependency collection of illustrative plates fails to detect nasopharyngeal carcinoma Disease-causing gene δ 6p21.3 (HLA) district (RR: the lower bound 20-upper bound is unlimited), and the common linkage analysis of haplotype (haplotype sharing linkage analysis) can detect.This has pointed out another problem in this field: based on the tactful lack of resolution of non-coding (district), can not detect in any human common cancer even the strongest genetic correlation.
Known or suspection numerous disease is the polygene disease.Think that the multigenic disease really of most of diseases, single-gene disorder are exceptions.Because the mode (there are differences) of all genes of hereditary M ﹠ F genotype makes art methods identify that the gene that relates to multigenic disease becomes complicated.Therefore, though adopted the drafting gene map of prior art and the method for discovery gene to identify the gene with particulate inheritance mode and the gene that relates separately to disease, but still the more strong method of significant need is illustrated the related gene of complex disease.
Therefore, the purpose of one aspect of the present invention is the problem that overcomes or alleviate at least the prior art deficiency.Specifically, the objective of the invention is to utilize monoploid information that the method for more accurate gene mapping is provided.
The file that comprises in the application's book, minutes, material, install, quote article etc., purpose just is used for providing content of the present invention, do not hint or represent the basic component that any or all these materials have constituted the prior art before the every prior claims of the application time, or the common world knowledge relevant with the present invention in this area.
Summary of the invention
The present invention relates to provide the method for the clear and definite haplotype of object.The haplotype information that produces with methods described herein is more more accurate than the haplotype method that prior art can only provide deduction.Therefore, an aspect of of the present present invention provides the method for the clear and definite haplotype of detected object, the nucleotide sequence information that this method comprises the steps: to provide the monoploid element that separates basically of object and obtains this monoploid element.The applicant proposes, utilizing the monoploid element that separates basically to eliminate makes error inference or misleads the problem of inferring two or more locus phases (phase) in the haplotype, thereby can disclose the two or more genes that participate in the haplotype, can be not complicated because of the difference of mode of inheritance.Can guarantee strict cis phase dependency by the haplotype element that uses basic separation as parent material the inventive method of sequential analysis.
In a kind of embodiment of described method, make sequence information relevant with allelotrope.In the another kind of embodiment of described method, described allelotrope is the allelotrope of encoding sequence.
The applicant recognizes in clear and definite haplotype, unites the importance of utilizing diploid material and maximum likelihood algorithm problem itself.Mistake in the expectation prior art method therefor is given birth to footling presentation the problem of particularly important when investigation has the proterties on polygene basis (disease).
In a kind of embodiment of described method, the step of separate object monoploid element comprises that employing diploid material is as the source of monoploid element basically.Can adopt any method well known to those skilled in the art or their combination, separate the monoploid element in diploid gene group or its part.In one embodiment of the invention, adopt physical method, as microdissection.For example, but micro-dissection cut karyomit(e) by the kinetochore and produce two chromatids.
Separable diploid cell is as somatic diploid gene group or a genomic part.Can avoid adopting natural monoploid material such as spermoblast and ovum, because obtain these sexual cell existing problems clinically.When considering that the reduction division process recombinates sometimes, when becoming trans connection as the locus that originally connects with cis, in the monoploid somatotype, avoid adopting gamete that a kind of advantage is arranged in addition.Therefore, the haplotype of analyzing gamete with analyze monoploid element available from diploid cell and may produce different (i.e. mistake) haplotype information.
The present invention's content on the other hand is to utilize the monoploid element to come the clear and definite haplotype of determination object.
A further aspect of the present invention provides the method for measuring dependency between gene region and the proterties, this method comprises the steps: to provide first group of monoploid element of a plurality of objects, the all individualities of described object have represented conventional crowd's genetic diversity, analyze described first group of monoploid element and whether have allelotrope; Second group of monoploid element of a plurality of objects of this routine crowd is provided, described object has (disease) proterties, described object is not from same family, analyze described second group of monoploid element and whether have allelotrope, measure total allelic level between first group and the second group of monoploid element; (disease) proterties is relevant therewith to exist excessive total allelotrope to show this equipotential gene.In a kind of embodiment of this method, described allelotrope is encoding sequence allelotrope.
The present invention provides the method for identifying its related gene to multigenic disease or proterties on the other hand, and this method comprises the employing methods described herein.Provide clear and definite haplotype described herein to eliminate and set forth chaotic uncertain that whether certain gene participates in the multigenic disease system.
The accompanying drawing summary
Fig. 1 shows that the laser pressure of human chromosome 6 launches the light micrograph of (catapulting).Each component from left to right is: (catapult) rearward vision is identified and launched to diffusion metaphase, karyomit(e) 6.
Fig. 2 shows the light micrograph that another routine human chromosome 6 launches.Left side figure identifies that right figure is for launching rearward vision.
Fig. 3 shows the nested PCR amplicon of the CFTR exons 10 that karyomit(e) 7 launches.Swimming lane 1-3 is karyomit(e) 7.Swimming lane 4 negative contrasts.Swimming lane 5 positive contrasts.
Fig. 4 shows the direct nested PCR product of HLA-A exon 2 among the individual chromosome 6s that laser pressure launches.Swimming lane 1 is that the laser pressure of individual chromosome 6 in 10 microlitres, 0.1% triton x-100 (Triton X-100) solution launches.Swimming lane 2 is that the laser pressure of individual chromosome 6 in 10 microlitres, the 0.1% triton x-100 solution launches.Swimming lane 3 is that the laser pressure of individual chromosome 6 in 10 microlitres, the 0.1% triton x-100 solution launches.Swimming lane 4 is that the laser pressure of individual chromosome 6 in 10 microlitres, the 0.1% triton x-100 solution launches.Swimming lane 5 is that the laser pressure of individual chromosome 6 in 10 microlitres, the 0.1% triton x-100 solution launches.Swimming lane 6 is that the laser pressure of individual chromosome 6 in 10 microlitres, the 0.1% triton x-100 solution launches.Swimming lane 7 is that the laser pressure of individual chromosome 6 in 10 microlitres, the 0.1% triton x-100 solution launches.Swimming lane 8 is that the laser pressure of individual chromosome 6 in 10 microlitres, the 0.1% triton x-100 solution launches.Swimming lane 9 negative contrasts, catch with 10 microlitres, 0.1% triton x-100 solution contained pen film clear area.Swimming lane 10 is for only containing the negative control of 10 microlitres, 0.1% triton x-100 solution.Swimming lane 11 positive contrasts, cumulative volume are to contain 1 microlitre 200pg/ μ l gDNA in the 0.1% triton x-100 solution of 10 microlitres.Swimming lane 12 is molecular weight marker VIII. Swimming lane 1,5 and 6 is used to order-checking. Swimming lane 1,5 and 6 is that pure 03 sequence-ring is very bright.
Fig. 5 shows the order-checking (result) to Fig. 4 swimming lane 1,5 and 6 HLA-A amplicon.
Fig. 6 shows the nested PCR amplicon of the DRB1 exon 2 that metaphase, laser launched.
Above two clotting glue (drawing together with being marked with the bracket of " single metaphase ") show DRB1*01 specific PCR (product).Above the swimming lane content of two clotting glue as follows: swimming lane 1 is molecular weight marker.Swimming lane 2 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 3 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 4 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 5 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 6 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 7 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 8 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 9 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 9 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 10 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 11 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 12 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 13 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 14 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 15 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 16 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 17 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 18 is mark.Swimming lane 19 is mark.Swimming lane 20 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 21 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 22 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 23 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 24 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 25 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 26 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 27 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.The positive contrast of swimming lane 28 (clear band), cumulative volume are to contain 1 microlitre 400pg/ μ l gDNA in the 0.1% triton x-100 solution of 10 microlitres.Swimming lane 29 is mark.
Below two clotting glue show DRB1*09 specific PCR (product).Swimming lane 1 is mark.Swimming lane 2 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 3 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 4 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 5 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 6 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 7 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 8 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 9 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 10 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 11 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 12 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 13 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 14 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 15 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 16 (clear band) is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 17 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 18 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 19 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.Swimming lane 20 is the single LMPC metaphase of catching with 6 microlitres, 0.1% triton x-100 solution.Swimming lane 21 is for containing the negative control of 6 microlitres, 0.1% triton x-100 solution.The positive contrast of swimming lane 22 (clear band), cumulative volume are to contain the pure gDNA of 1 microlitre 1ng/ μ l in the 0.1% triton x-100 solution of 10 microlitres.Swimming lane 23 is mark.
Fig. 7 shows that karyomit(e) launches the Dot blot sign of the DRB1 amplicon of (chromopult) generation.
Detailed Description Of The Invention
One aspect of the present invention content provides the method for the clear and definite haplotype of determination object, the nucleotide sequence information that this method comprises the steps: to provide the monoploid element that separates basically of object and obtains this monoploid element.The applicant proposes, utilizing the monoploid element that separates basically to eliminate makes error inference mutually or misleads the problem of inferring two or more locus in the haplotype, thereby can disclose the two or more genes that participate in the haplotype, can be not complicated because of the difference of mode of inheritance.
Estimate that the present invention has improved prior art and detected haploid method, this paper has described clear and definite monoploid classifying method.In order to understand their difference better, should consider that haplotype concept that present this area is understood and the method for determination object haplotype set forth again.
Term " haplotype " is the simplification appellation of " monoploid genotype " phrase, its implication that is accepted at present be exist on mother or the father's individual chromosome usually as one group of nucleotide sequence polymorphism or the allelotrope of unit heredity together.With diplont such as artificial example, the haplotype of certain locus contains a member in the pair of alleles.
In the method for prior art, the mensuration of haplotype begins to adopt from the conventional diploid material that obtains clinically.The applicant proposes, and the embodiment of accepting that this employing diploid material carries out the monoploid somatotype is not enough.Another kind method is to adopt natural monoploid material (for example sperm or ovum), yet this is generally very inconvenient, and the women will suffer (operation) excessively invasion.In addition.The sequence information that gamete provides is hybridized and the meeting confusion reigned as the cis phase dependency between the SNP owing to what take place during the reduction division, can not guarantee reliably.
Because the cis that adopts the method for diploid DNA can not directly measure as haplotype connects locus, so can adopt maximum likelihood and similar occurrence probability of inferring haplotype based on the estimation of algorithm.Therefore think always that two kinds of polymorphisms that cis may exist in being connected can be as Mendelian unit heredity together.Therefore the more accurate description of this method of prior art should be " haplotype of deduction ".The inventive method thinks that the fundamental unit of Mendelian inheritance is is the chromosome segment on border with father's recombination site, thereby more near the explication of haplotype.
Therefore, those skilled in the art can not suspect whether to produce the problem of utilizing the diploid raw material to produce erroneous effects.Therefore also do not recognize mensuration crowd correlated character, or the issuable problem of object haplotype.On the contrary, the applicant recognizes the importance of utilizing diploid material and the next clear and definite haplotype problem of maximum likelihood algorithm itself uniting.Estimate that the mistake in the prior art method therefor will produce the problem of particularly important when investigation has the proterties on polygene basis (disease).
Therefore, the present invention relates to measure the method for clear and definite haplotype.Term used herein " clear and definite haplotype " refers to only to think (haplotype) that strict cis is relevant when the attribution haplotype.Methods described herein comprise any estimation or deduction, no matter whether there are two kinds of polymorphisms on the same dna molecular.These are different with above-mentioned " haplotype of deduction " that obtains by the sequence information (as implementing in the HapMap scheme) of inquiry diploid material.
Except the problem that infer to produce, utilize diploid cell also can introduce complicacy (" allelotrope withdraws from (allele dropout) ") due to the short allelotrope advantage, this moment can not the bigger allelotrope of specific amplification.Therefore, bigger allelic existence and the wrong haplotype information of generation may have been ignored fully.
The monoploid element that the inventive method utilization separates has basically guaranteed the dependency (analysis) of strict correct cis phase as the raw material of sequential analysis.Term used herein " monoploid element " refers to comprise to have only that mother produces or have only any nucleic acid molecule (DNA, RNA or derivatives thereof) of the nucleotide sequence that father produces.This monoploid element can be the nucleic acid molecule of any length, comprises the part of total length karyomit(e), chromatid or chromatid.The present invention utilizes one or more monoploid elements to interrogate sequence information.For the mankind, described method adopts 46 all monoploid elements and analyzes 46 all monoploid elements respectively.
Term " separates basically " and refers to be substantially free of the pollution genetic material of only monoploid element cis phase dependency being identified in the time of might disturbing query.Usually when thinking that this monoploid element is father when producing, the genetic material of its pollution is the monoploid element that mother produces; When thinking that this monoploid element is father when producing, the genetic material of its pollution is the monoploid element that mother produces.
In one embodiment of the invention, described method comprises the haploid step of separate object basically.Obtaining basically, the method for the monoploid element of separation can be any appropriate method well known by persons skilled in the art.Should understand realization and separate basically and be not to limit the scope of the invention, but in a kind of embodiment of described method, comprise with physical method and separate this kind monoploid element basically.But should be understood that the pollution genetic material in the physical property removal monoploid element, the monoploid element is separated with the genetic material of pollution.When having the monoploid element in the diploid cell, can adopt this method.For example, the manual operations or carry out microdissection by contactless instrumentation and obtain this kind monoploid element.Perhaps remove pollution genetic material in this kind monoploid element with physical method in when query.
In the another kind of embodiment of described method, the genetic material that deactivation or excision are polluted makes it no longer have the function of polluting genetic material.For example, this can utilize the laser beam irradiation of careful guiding to destroy homologous chromosomes and realize.
Another kind of possible method is with this monoploid element of PCR selective amplification, makes the copy number of monoploid element DNA substantially exceed the DNA of pollution.Available nuclease partly digests this dna molecular mixture then, and the DNA of all pollutions is removed in digestion basically, and stays low-level monoploid element.
Also can pass through long PCR (long PCR), adopt this monoploid element of primer selective amplification that has added label, utilize this label to separate then and obtain monoploid element copy.
This monoploid element in separable object genome or the genome part.Term used herein " genome " refers to the full gene material of object, comprises a complete set of dna sequence dna of object.Genomic " part " refers to the part of nucleic acid, for example the genomic thymus nucleic acid of object (DNA).
Cell be can obtain or genome or its part in the biological nucleic acid sample contained.When described genome during available from diploid cell, can induce this cell to enter metaphase by adding inductor well known by persons skilled in the art such as Colchiceinamidum.Metaphase occurs discontinuous karyomit(e), can as described below its dissection be become the monoploid element.
In a kind of embodiment of described method, the step of the monoploid element of separate object comprises and utilizes the diploid material as the source of this monoploid element basically.Can adopt any method well known to those skilled in the art or integrated processes to separate the monoploid element that obtains in diploid gene group or its part.The present invention also comprises any other method that may develop in the future.In one embodiment of the invention, adopt physical method such as microdissection.Can cut off chromosomal kinetochore by micro-dissection and produce two chromatids, each is the monoploid element.Perhaps, produce p and q arm in kinetochore distal severed chromosome segregation, each arm is the monoploid element.Therefore, this monoploid element can be chromatid or chromatid section.
The technician is familiar with being used for micrurgic platform and instrument.Though technical strict, comprise available micro-dissection equipment in the Advanced Concepts Laboratory equipment.Equipment Requirement comprise be furnished with micromanipulator and universal stage, move the liquid aspirator microscope of (can produce micro-needle).Recommendation has the microscope of vibratory separator.Though do not need the laboratory of special cleaning, the chromosome segment that micro-dissection obtains only contains the DNA of femtogram amount, must control it and be polluted by exogenous DNA.
Can adopt contactless method.An example of this method is to adopt laser microbeam.The laser microbeam micro-dissection comprises the pulsed Ultra-Violet Laser of the high beam quality (beam quality) of employing and microscope interfaces.German P.A.L.M. company (P.A.L.M.GmbH Bernried, Grmany) P.A.L.M of Zhi Zaoing that can adopt that market buys
Robert's Microbeam Instrument (P.A.L.M
Robot Microbeam) carries out the laser beam micro-dissection.The genome section can not damaged or destroy to described Wavelength of Laser usually, as the maximum absorption wavelength wide apart of 337nm laser freestone acid as DNA.
Another system that can be used for the dissection of karyomit(e) laser capture microdissection is Lycra laser capture microdissection dissecting microscope.This system adopts the DMLA upright microscope, comprises microscopical all other advantages of removable nozzle, moveable platform, xyz-controlling elements and novel DMLA.Used laser is the Ultra-Violet Laser of wavelength 337nm.By mobile irradiates light cutting, keep platform static simultaneously.Mark area-of-interest at monitor, with PC control cutting.Make sample fall into the PCR test tube without additional force.Be not difficult to verify the result of cutting by the automatization mode of checking.
For example, available PALM laser utilizes laser to launch karyomit(e) and carries out micro-dissection, realizes the separation of monoploid element.At this moment, described noncontact mode comprises the periphery with laser ablation target monoploid element, launches with laser pressure then to make determined element fall into the test tube lid, covers the DNA of the single arm of subsequent analysis as micro-centrifuge tube.
Available laser pressure launches and reclaims the monoploid element that produces.Realize that in (for example) interested monoploid genome section or several segments laser pressure launches by the laser focusing microbeam, generation power causes required monoploid element to launch unwanted genome section owing to the photon density height that produces.In a kind of embodiment of described method, make sample mobile and launched in the collection tube above light wave.Those skilled in the art will know that suitable collection tube, comprise for example polymerase chain reaction (PCR) test tube or micro-centrifuge tube commonly used.
Available preparation type flow cytometer separates acquisition monoploid element basically.Other method is to adopt the radiation-like crossbred, wherein the diploid material of Fa Yuing comprise human chromosome as unique each to one of karyomit(e).Another strategy is " the genetic modification technology " that adopts GMP Science and Technology Ltd. (GMP Technologies Inc.) exploitation.GMP genetic modification technology (GMP Conversion Technology)
The method that adopts can become individual chromosome with paired chromosome segregation.After the separation, the gene probe of available energy identified gene sequence is analyzed allelotrope respectively.This technology can be applicable to gene, karyomit(e) or whole human genome.
Usually the autosomal diploid gene group of chorista cell or its a part of term " euchromosome " any karyomit(e) except sex chromosome in normal somatocyte or the sexual cell of making a comment or criticism.For example, people's 1-22 karyomit(e) is exactly euchromosome.
As mentioned above, avoided adopting natural monoploid material such as spermoblast or ovum to be owing to obtain these sexual cell existing problems clinically.Avoiding adopting another advantage of gamete in the monoploid somatotype is may recombinate sometimes when should be taken into account reduction division, and the locus that original cis is connected becomes trans relevant.Therefore, the haplotype of analyzing gamete will produce different (i.e. mistake) the haplotype information of shop times body member with the diploid cell acquisition.
Refer to comprise the nucleotide sequence of measuring nucleic acid molecule with any method well known by persons skilled in the art referring to " acquisition sequence information ", comprise direct order-checking.The available technology of knowing, " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual) of writing such as Maniatis for example, cold spring harbor laboratory (Cold Spring Habor Laboratory), those technology described in 1982 obtain nucleotide sequence.Also available automatic technology comprises that the instrument that utilization is bought from market carries out nucleic acid sequencing.Term used herein " nucleic acid " comprises strand or double chain acid molecule, comprises fragment or the part of complete nucleic acid molecule.Comprise DNA and RNA the two.
Also available indirect method obtains sequence information as adopting oligonucleotide probe.Obtain sequence information and generally include evaluation SNP, those skilled in the art have the ability to design the probe that can detect SNP.Probe is long approximately 25 Nucleotide generally, are positioned at probe core during the polymorphic position dot blot of design.
The precursor step that obtains sequence information normally has the primer amplification nucleic acid of area-of-interest with side joint.Can obtain the DNA in any parenchymal tissue source (except pure red corpuscle).For example, the tissue sample that can conveniently obtain comprises whole blood, seminal fluid, saliva, tears, urine, fecal material, sweat, mouthful cheek liquid, skin and hair.
The DNA of preparation can use proper method well known by persons skilled in the art, comprises and adopts the PCR method of corresponding primer to analyze.When needs are analyzed whole genome, can adopt whole genome amplification (WGA) method.For example, but trans-activation postmitotic cell and separate and obtain two kinds of monoploid carries out WGA then.The used commercial kit of this kind method is not difficult to buy, and comprises St. Louis Sigma company (Sigma-Aldrich Corp), St Louis, MO, GenoPlex USA)
Complete WGA test kit (GenoPlex
Gomplete WGAkit).This test kit becomes a series of templates based on genomic random fragmentation.The shorter dna chain that obtains can produce and contain the dna fragmentation storehouse that 3 ' end causes and 5 ' end causes.Copy this storehouse at initial period with linear constant-temperature amplification, use how much (PCR) amplification of limited round then.
With pcr amplified dna many methods are arranged.These methods comprise that (PNAS 96 (8): 4494-4499,1999-4-13) described (1) does not have the deflection whole genome amplification to people such as Klein fully, is the tens of methods to hundreds of locus in the monoploid element that separates that can increase with (2) prospect.
Also often the mRNA sample is increased.At this moment, want reverse transcription before the amplification usually.Can be by the mRNA of all expression of amplification described in WO96/14839 and the WO 97/01603.If sampling to as if heterozygote in the mRNA that expresses, contain pleomorphism site, the RNA in its diploid sample that increases can produce two kinds of target molecules.
The method that makes things convenient for of identifying nucleotide polymorphisms is to adopt microarray technology.This kind of the present invention enforcement example generation after generation can be referring to Affymetrix
Gene chip (the GeneChip that puts on market
) technology.This technical basis photoetch processing 5 " * 5 " the quartz wafer coatings, and the photoactivation compound prevents between first Nucleotide of wafer and dna probe coupling taking place.Utilize planography tinted shade blocking light or transmit light to the specific position of wafer surface.This surface is immersed in the solution that contains adenosine, thymidine, cytidine or guanosine, and coupling only occurs on the slide in those zones by the illumination deprotection.The nucleic acid of coupling also has the protection against light sensitivity group, therefore wants recirculation to carry out.Many companies, (the Oxford Gene Technology of Oxford Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080 that comprises England Oxford, Oxford, UK), the A Jilante scientific ﹠ technical corporation of California, USA Paro atropic (Agilent Technologies, Palo Alto, CA USA) and the Ning Bulai root system of Wisconsin, USA Madison system (the Nimblegen Systems Inc. of company limited, Madison, WI USA) provides the method for other stationary probe.
Two kinds of haplotype information of the object that expectation can provide designed probe join on the micro-array chip.Be responsible for providing the probe of father's haplotype information can be with one type fluorescence labels mark, and mother's haplotype information can be with different labels.
In one embodiment of the invention, two or more polymorphisms have been formed the part of allelotrope encoding sequence.Available the inventive method is by proving the excessive dependency that shows between gene and the proterties of total allelotrope.In the content of the present invention, term " allelotrope " is used in reference to the genovariation relevant with the coding region, comprises the alternately splicing form of given gene.Term used herein " total allelotrope " refers to that certain gene exists in one group of individuality (or sharing) allelotrope.One of getable many statistics software packages of techniques available personnel are found total allelic existence and degree.Utilize the used non-coding sequence variant (as SNP and STR) of (having) and HapMap allelotrope encoding sequence inequality can show the haplotype that has coding indirectly.This HapMap indirect method itself can not determine SNP whether represented the length of the diversity of colony's haplotype, haplotype parts, linkage disequilibrium with mutually.Though the invention is not restricted to analyze any specific allelotrope, exemplary allelotrope is those allelotrope relevant with HLA-A with CFTR, DRB1.
The present invention provides the clear and definite haplotype that utilizes the monoploid element to come determination object on the other hand.
A further aspect of the present invention provides the method for measuring dependency between gene regions and the proterties, this method may further comprise the steps: first group of monoploid element that a plurality of objects are provided, described individuality has represented overall crowd's genetic diversity, analyze in this first group of monoploid element and whether have allelotrope, second group of monoploid element of a plurality of objects of overall crowd is provided, described object has proterties and is not from same family, analyze in this second group of monoploid element and whether have allelotrope, measure between first and second group monoploid element total allelic level in the allelotrope, total allelotrope is excessive shows that this equipotential gene is relevant with proterties.In a kind of embodiment of this method, described allelotrope is encoding sequence allelotrope.
The present invention provides the method for identifying the gene that relates to multigenic disease or proterties on the other hand, and this method comprises the method as herein described that adopts.Infer in view of existing monoploid classifying method relies on, be not impossible but be very difficult to find out fully certain given gene effect in multigenic disease or proterties.And clear and definite haplotype method provided herein can be eliminated the confusion of the individual gene that relates in the polygenic system disease is set forth the uncertainty that produces.
It will be understood by those skilled in the art that the present invention can have many purposes.In one embodiment, the invention provides the gene that utilizes methods described herein to identify to relate to single-gene disorder or phenotype.Another embodiment is identified the gene that relates to multigenic disease or phenotype with methods described herein.Provide clear and definite haplotype also can tissue transplantation the time donor and receptor better mate.
The present invention can be applicable to any object.Described object can be people, horse, ox, goat, sheep, dog, cat or pig.The technician will understand, and will be biological as plant also suitable this application.Some biology is polyploid (as certain plants and the shell hydrobiont arranged), and in view of there is three or more homologous chromosomes in their each cell and the chaotic situation that produces, expection the present invention have even bigger advantage.
A further aspect of the present invention provides the gene of identifying the participation drug reaction with methods described herein.Pharmacogenomics is that the individual hereditary property of research is how to influence body to the reaction of medicine.The basis of pharmacogenomics is to can be individuality to customize medicine by their genomic constitution of itself.Environment, diet, age, life type and state of health etc. all may influence individual reaction to pharmacological agent, are the keys of formulating more efficient safer individual administration but should understand the genomic constitution of thinking individual.
Utilize the inventive method, can be according to formulating dosage regimen with protein, enzyme and the RNA molecule of gene and disease-related.This will be conducive to find medicine, make the researchist can formulate the treatment plan of target specific ill (cell), and its levels of precision has not only at utmost improved curative effect but also reduced closing on the damage of healthy cell.
The repetition test method that patient and correct medicine are matched of replacement standard, the clinicist can analyze patient's hereditary general picture, thereby can leave best available medicine prescription when the treatment beginning.This not only can guess and the correct medicine that will determine for patient, and has accelerated rehabilitation duration and improved safety owing to having eliminated the possibility that untoward reaction takes place.What the potential remarkable reduction of the pharmacogenomics U.S. estimated causes the present situation that annual 100000 people of generation are dead and 2 million peoples are in hospital because of adverse drug reaction.
Implementing a kind of the possibility of result of the present invention is to make the method that more accurate mensuration medicine is fit to dosage.At present determine that according to body weight and age the method for basal dose will replace the genetic dosage method according to patient.According to accurate hereditary general picture, for example how time of spending of this medicine of metabolism and this medicine of katabolism is formulated dosage regimen according to body.This will at utmost improve curative effect and reduce excessive possibility.
Implementing another kind of the possibility of result of the present invention is to improve the disease method for screening.The hereditary general picture of aware of individual just may clear and definite its susceptibility to one or more diseases.Knowing this susceptibility then can allow the individual take correct mode of life when younger and change environment and inherited disease takes place or alleviate its severity avoiding.Equally, the susceptibility of knowing in advance specified disease can carefully monitor this individuality, treats in the optimal stage and makes curative effect the best.
Implement of the present inventionly also have a kind of result to improve vaccine inoculation.With the vaccine of genetic material such as DNA or RNA preparation not now with vaccine dangerous and demonstrate good application prospect.Gene vaccine can activating immune system but is not caused infection.This kind vaccine is not expensive, stablize, be easy to store, can be through engineered and carry (antigen) of several pathogenic agent simultaneously.
Also can utilize the present invention to accelerate drug development and examination and approval procedures.Drugmaker utilizes the target sequence in the genome can the potential medicine of easier discovery.Can look back in the past failure drug candidate they whether can with profit market personnel's faciation coupling of its service.Should be able to accelerate the drug approval process, because the clinical trial of declaring is at specific genetic diseases crowd, as long as successful degree is better.By only carrying out clinical trial at those people that medicine had reaction can reduce expense and danger.
Therefore, accurate genetic information provided by the invention can reduce adverse drug reaction generation, reduce clinical drug trial failure, reduce authorities' approval medicinal application time, reduce patient treatment time, reduce patient and obtain medication amount that effective treatment must take, reduce the target disease scope that disease may be treated the influence (passing through early detection) of body and raising medicine.The clean reduction that these advantages can cause health subsidies to be used.
As mentioned above, the inventive method also can be used for identifying gives individual gene or a plurality of gene to disease-susceptible humans, and example specifically is multigenic disease such as diabetes.For example, the possibility that the sick tumor susceptibility gene of the most of glycosurias that have been found that makes the people who has the defective copy that diabetes B take place has improved 3 times.SNP in known calpain-10 (a kind of proteolytic enzyme) gene is relevant with diabetes B.Easily suffer from this sick Mexico American and proved this dependency.To the order-checking of this crowd's DNA sample and carry out statistical analysis, find that these Mexico American has insulin resistance, show that calpain-10 expression level reduces.The present invention can more simplify the mensuration of gene/disease-related, promotes the gene basis of other heredity complex disease such as asthma, schizophrenia and degenerative brain disorder to identify.
The present invention is provided for identifying medicine target gene (or target protein) or gene therapy methods on the other hand.Implement the present invention and can understand the hereditary basis of disease, thereby effective target of medicinal design and gene therapy is provided.For example, if identify certain gene or some gene and disease-related, the activity that can strengthen or suppress this gene provides result for the treatment of.Perhaps, can utilize the protein product of this gene to carry out the screening test as the basis, by the activity in conjunction with this albumen of adjusting.In addition, if understand the three-dimensional structure of this albumen, can carry out rational medicinal design.
On the other hand, the invention provides with the individuality of methods described herein evaluation to disease-susceptible humans.In case it is relevant with specified disease to identify certain gene, adopt the inventive method technician can design the test of this genetic flaw form of screening.Identifying the individual who is in some disease incidence danger can take preventive measures, as give pharmacological agent and change lifestyles.
Also have on the one hand, the invention provides with described method evaluation environment is irritated easy aitiogenic individuality.An example of this application is the individual who identifies various material allergy.Some individual may cause potential lethality anaphylaxis to the reaction of anaphylactogen (as peanut or bee venom).Identify responsive especially individuality and can take the deallergization treatment procedure.
Now by following non-limiting example the present invention is described.
Embodiment
Embodiment 1: utilize laser capture microdissection dissection and pressure (LMPC) program of launching to carry out chromosome segregation metaphase under the 100X object lens with PALM Robert Microbeam Instrument
At slide glass preparation cell smear metaphase, this slide glass is fit to carry out laser capture with the PALM microscope under the 100x object lens subsequently.With one metaphase karyomit(e) and its sister chromosome make spatial isolation, comprise with standard P .A.L.M microscope program individual chromosome launched in the cap of the flat cap PCR pipe of the overflux of the 200 μ l that 6 microlitre 0.1% (v/v) triton-X-100 are housed (UltraFlux Flat Cap PCR tube).Centrifugal making launched thing and transferred to the pipe end and be used for to analyze.
Embodiment 2: the karyomit(e) that amplification separates
Catch separated DNA by PCR or MDA with standard program amplification laser capture microdissection.The exon specific PCR amplification program of CFTR (exons 1 0), HLA-A (exon 2), DRB1 (exon 2) is as follows.
CFTR
With the cftr gene seat exons 10 in nested PCR method amplification gDNA, metaphase or the individual chromosome.First round amplified reaction volume is 30 microlitres, adopts the Taq archaeal dna polymerase, and the primer is as follows:
CF-1F 5 '-GACTTCACTTCTAATGATGAT-3 ' and
CF-1R 5′-CTCTTCTAGTTGGCATGC-3′
Cycling condition is as follows: 95 ℃ 3 minutes; 95 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 45 seconds, totally 25 take turns; 72 ℃ 5 minutes; 4 ℃ of maintenances.
Carry out second with first round product as template and take turns nested PCR, the primer is as follows:
CF-2F 5 '-TGGGAGAACTGGAGCCTT-3 ' and
Cf-2r 5′-GCTTTGATGACGCTTCTGTAT-3′
2 microlitre first round products are transferred in the 30 microlitre reaction solutions, adopted the Taq polysaccharase, cycling condition is as follows: 95 ℃ 3 minutes; 95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, totally 40 take turns; 72 ℃ 5 minutes; 4 ℃ of maintenances.
HLA-A
With the HLA-A locus exon 2 in nested PCR method amplification gDNA, metaphase or the individual chromosome.First round amplification HLA-A exon 2 adopts following universal primer:
SQR2
5 '-CTCGGACCCGGAGACTGT-3 ' and
M13_5AIn1-46
5′-TGTAAAACGACGGCCAGTGAAACSGCCTCTGYGGGGAGAAGCAA-3′
Genomic dna, single metaphase or individual chromosome are used as starting template.PCR adopts Platinum Taq archaeal dna polymerase, and reaction volume is 25 microlitres, reacts under following cycling condition: 98 ℃ 2 minutes; 98 ℃ 5 seconds, 60 ℃ 120 seconds, 72 ℃ 120 seconds, 10 take turns; 98 ℃ 5 seconds, 65 ℃ 30 seconds, 72 ℃ 60 seconds, 25 take turns; 72 ℃ 10 minutes, 4 ℃ of maintenances.
Carry out second of HLA-A locus exon 2 with 2 microlitre first round products and take turns nested type amplification, reaction volume 25 microlitres, cycling condition is as follows: 98 ℃ 30 seconds; 98 ℃ 5 seconds, 65 ℃ 30 seconds, 72 ℃ 60 seconds, 10 take turns; 98 ℃ 5 seconds, 60 ℃ 30 seconds, 72 ℃ 60 seconds, 25 take turns; 72 ℃ 10 minutes; 4 ℃ of maintenances.Adopt following primer:
AFE2174G 5 '-GGGACCTGCAGACACGGAATG-3 ' and
HLA-DRB1
With the HLA-DRB1 locus exon 2 in the amplification of nested PCR method gDNA, metaphase or the individual chromosome.With Auele Specific Primer carry out HLA-DRB1* 01 and-amplification of first round of DRB1*09 exon 2.Adopt following primer amplification DRB1*01:
RB1mf
5 '-TGTAAAACGACGGCCAGTTCCCAGTGCCCGCTCCCT-3 ' and
RB2mr
5′-CAGGAAACAGCTATGACCACACACTCAGATTCTCCGCTT-3′
Adopt following primer amplification DRB1*09:
I1-RB15mf
5 '-TGTAAAACGACGGCCAGTCAGTTAAGGTTCCAGTGCCA-3 ' and
I2-RB28mr
5′-CAGGAAACAGCTATGACCACACACACACTCAGATTCCCA-3′
Genomic dna, single metaphase or individual chromosome are used as starting template.PCR adopts Platinum Taq archaeal dna polymerase, and volume 25 microlitres react under following cycling condition: 95 ℃ 3 minutes; 95 ℃ 30 seconds, 50 ℃ 120 seconds, 72 ℃ 90 seconds, 30 take turns; 72 ℃ 10 minutes, 4 ℃ of maintenances.
50 microlitres second are taken turns and are comprised this first set reaction liquid of 2 microlitres in (nested) reaction solution, adopt Taq archaeal dna polymerase and degraded primer:
Cycling condition is as follows: 95 ℃ 3 minutes; 95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, 30-40 wheel; 72 ℃ 5 minutes; 4 ℃ of maintenances.
The analytical results of cftr gene seat 10, HLA-A, HLA-DRB1 is seen Fig. 1-7.These figure have confirmed to dissect and catapult technique has obtained the valuable sequence information of the monoploid element that separates with laser capture microdissection.
At last, should know, can make various other modifications and/or variation to (foregoing), but not break away from thinking of the present invention as herein described.
Claims (12)
1. the method for the clear and definite haplotype of a determination object, this method comprises the steps:
I) karyomit(e) or its fragment metaphase of separate object; With
Ii) obtain described metaphase of karyomit(e) or the nucleotide sequence information of its fragment;
Wherein, described clear and definite haplotype includes the nucleotide sequence information available from described metaphase karyomit(e) or its fragment.
2. the method for claim 1 is characterized in that, described method comprises with laser launches step I) in described metaphase of karyomit(e) or its fragment.
3. method as claimed in claim 1 or 2 is characterized in that, described object metaphase karyomit(e) or its fragment separate from the diploid material.
4. method as claimed in claim 3 is characterized in that, described diploid material is available from somatocyte.
5. method as claimed in claim 1 or 2 is characterized in that, described metaphase, chromosomal fragment was a section of chromatid or chromatid.
6. as each described method among the claim 1-5, it is characterized in that whether described nucleotide sequence information exists single nucleotide polymorphism.
7. as each described method among the claim 1-6, it is characterized in that described sequence information provides by direct order-checking.
8. as each described method among the claim 1-6, it is characterized in that described sequence information is by providing with oligonucleotide probe hybridization.
9. method as claimed in claim 8 is characterized in that, described oligonucleotide probe hybridization represents to exist single nucleotide polymorphism.
10. method as claimed in claim 9 is characterized in that, only provides cis phase single nucleotide polymorphism relevant.
11., it is characterized in that described sequence information is about coding region allelotrope as each described method among the claim 7-10.
12., it is characterized in that described sequence information is about CFTR, DRB1 or HLA-A locus as each described method among the claim 7-11.
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| AU2005906773A AU2005906773A0 (en) | 2005-12-02 | Methods for gene mapping and halotyping | |
| PCT/AU2006/001836 WO2007062486A1 (en) | 2005-12-02 | 2006-12-04 | Methods for gene mapping and haplotyping |
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| US5972614A (en) * | 1995-12-06 | 1999-10-26 | Genaissance Pharmaceuticals | Genome anthologies for harvesting gene variants |
| US6228587B1 (en) * | 1999-03-08 | 2001-05-08 | Xin-Yuan Guan | Chromosome microdissection method |
| WO2001083691A2 (en) * | 2000-04-12 | 2001-11-08 | The Cleveland Clinic Foundation | System for identifying and analyzing expression of are-containing genes |
| EP1246114A2 (en) * | 2001-03-30 | 2002-10-02 | Perlegen Sciences, Inc. | Methods for genomic analysis |
| CN1646701A (en) * | 2002-01-30 | 2005-07-27 | 波里尔植物培育有限公司 | Method and test kit for demonstrating genetic identity |
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|---|---|---|---|---|
| US5972614A (en) * | 1995-12-06 | 1999-10-26 | Genaissance Pharmaceuticals | Genome anthologies for harvesting gene variants |
| US6228587B1 (en) * | 1999-03-08 | 2001-05-08 | Xin-Yuan Guan | Chromosome microdissection method |
| WO2001083691A2 (en) * | 2000-04-12 | 2001-11-08 | The Cleveland Clinic Foundation | System for identifying and analyzing expression of are-containing genes |
| EP1246114A2 (en) * | 2001-03-30 | 2002-10-02 | Perlegen Sciences, Inc. | Methods for genomic analysis |
| CN1646701A (en) * | 2002-01-30 | 2005-07-27 | 波里尔植物培育有限公司 | Method and test kit for demonstrating genetic identity |
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