CN105385755A - Method for conducting SNP-haplotype analysis by means of multiplex PCR technology - Google Patents
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Abstract
The invention discloses a method for conducting SNP-haplotype analysis by means of a multiplex PCR technology. Designed primers are combined into one tube for single primer amplification when synthesis is conducted, and meanwhile whole genome amplification is completed through an MALBAC technology; when multiplex PCR amplification is conducted, a touch down PCR amplification program is adopted, high-throughput sequencing and reasonable data analysis are combined, only one set of SNP detection is needed, different schemes do not need to be designed according to different objects, the working cycle is shortened, and the cost is reduced. In addition, according to the method for conducting the SNP-haplotype analysis by means of the multiplex PCR technology, family and embryonic genome SNP information is captured, embryo SNP information can be effectively and accurately determined, SNP-haplotype analysis is conducted to determine whether an embryo carries mutation sites of a genetic gene or not, and the defects that in the prior art, the technological cost is high, the cycle is long, and the application range is small are effectively overcome.
Description
Technical field
The present invention relates to molecular biology and field of bioinformatics, be specifically related to a kind of method utilizing multiple PCR technique to carry out SNP-haplotyping.
Background technology
World Health Organization's statistics in 2014, inborn defect affects 1 people greatly in 33 babies, causes the deformity that about 3,200,000 examples are relevant to inborn defect every year, estimates at 270,000 newborn infants every year and die from inborn defect.Inborn defect can be derived from the factor of heredity, infection or environment aspect, and many inborn defects can be prevented, such as single gene inheritance disease, carry out sufficient pregnant before and Prenatal Screening be crucial.
Diagnosis (PGD) technology refers in vitro in fertilization process before Embryonic limb bud cell, front biopsy and genetic analysis are implanted to the embryo with genetic risk patient, to select the Embryonic limb bud cell uterine cavity without genetic disease, thus obtain the diagnostic method of normal fetus, generation and the transmission of single gene inheritance disease can be blocked from root, the prevention of inborn defect is advanceed to embryo stage.But, the PGD of single gene inheritance disease detects not widespread use, and its reason, mainly due to specimen amount few (only several cell), easily produces allele dropout (ADO) and pollutes, detect comparatively difficulty, prior art cannot meet PGD testing requirement completely.
Before Embryonic limb bud cell, haplotyping is the main method of current PGD detection technique.The method, by detecting mutational site and multiple STR chain with it (STR) or single nucleotide polymorphism (SNP) determines mutation linkage haplotype, reduces the impact of amplified allele imbalance, ADO and pollution.Wherein, the haplotyping that the multiple STR of multiple fluorescence PCR combine with technique carries out mutational site once became the main technical schemes that PGD detects linkage analysis, but, STR linked marker is less, be applied in concrete case and may there is no available STR site, so need before carrying out clinical detection to do a large amount of preliminary experiment work.And STR genetic marker mostly distance pathogenic sites is comparatively far away, may cause mistaken diagnosis due to Chromosome recombination.
On the contrary, SNP extensively exists in human genome, 1 is just had in average every 500-1000 base pair (bp), estimate its sum can reach 3,000,000 even more, and the phenomenon such as multiple core sequences that SNP can not occur to exist in STR repeat, the non-multiple of core sequence repeats, in addition, SNP detection is lower to the requirement of DNA sample, price is more cheap.Therefore, this possess that density is high, quantity is many, stability is high and the SNP marker of the features such as high-throughput, replaces the first-selection that STR marks as linkage analysis gradually.
At present, detect genetic marker typing method widely usedly to mainly contain multiple fluorescence PCR (MF-PCR), DNA chip technology (SNP-array), utilize probe to catch SNP (international publication number WO2015/042980) etc., facilitate the development of SNP-haplotyping to a certain extent.But above-mentioned each technology all exists certain defect, such as multiple fluorescence PCR technology adopts multiplex PCR combined with fluorescent probe in detecting STR somatotype, because STR mark is less, distant easy restructuring, in practice, available STR site is less, testing cost is higher, so technology cannot carry out batch detection; And the method for SNP caught by chip and probe, compared with catching SNP method with regular-PCR, high expensive, the cycle is partially long.
Summary of the invention
Technical problem to be solved by this invention is exactly to overcome above-mentioned deficiency existing in prior art, and provide a kind of method utilizing multiple PCR technique to carry out SNP-haplotyping, it is simple to operate, result is accurate, can shorten sense cycle, reduce costs.
Object of the present invention is achieved through the following technical solutions: the invention provides a kind of method utilizing multiple PCR technique to carry out SNP-haplotyping, concrete steps are as described below.
1. according to target area design primer
1.1 determine target gene, according to concrete kind, with the particular location that NCBI (NationalCenterforBiotechnologyInformation) official website gene order is reference sequences determination target gene designation of chromosome, thus lock acquisition region.
1.2 multiplex PCR-SNP design
1.2.1 select SNP region, be defined within the scope of goal gene upstream and downstream 1M, heterozygosity higher (thousand parts of individual data items storehouse medium frequencys are greater than 0.3); SNP spacing is less, and recombination fraction is less.
1.2.2SNP areas captured scope, at SNP site upstream and downstream 100bp, catching clip size is about 200bp.
1.2.3 design of primers: utilize the primer-design softwares such as Oligo, Primer or primer Specialty Design website to carry out design of primers, the annealing temperature of each SNP site primer to be controlled during design in more among a small circle (as 50-55 DEG C, the scope such as 53-58 DEG C, 55-60 DEG C or 50-60 DEG C) as far as possible, be beneficial to pcr amplification below, primer length then controls at about 20-25bp.
1.2.4 primer assessment: adopt the professional websites such as NCBI or UCSC to carry out primer specificity assessment (as mispairing rate, hairpin structure etc.) after design of primers completes, assess qualified after carry out primer synthesis, all primers equimolar ratio example when synthesizing of design merges into a pipe, and the single tube that increases after utilization operates.
2. family sample and embryo's sample prepare
2.1 extract diseased individuals, the male parent of diseased individuals and the peripheral blood sample of female parent in family, obtain genomic dna.
2.2 embryo collection cell samples, utilize MALBAC technology to complete the whole genome amplification of embryonic cell; Wherein, embryonic cell can be selected from people or other Mammalss.
3. multiplexed PCR amplification, carries out catching of target area SNP to family and embryo's sample.
All primers of design in step 1 equimolar ratio example when synthesizing is merged into a pipe, and as single primer amplification, a pipe operation, increase dozens or even hundreds of SNP site simultaneously.Adopt touchdownPCR amplification program, cover multipair primer annealing temperature, improve amplification efficiency, finally can reach the coverage of 90%.
For the preliminary stage of multiplexed PCR amplification, need preliminary experiment be carried out, be optimized mainly through the following aspects:
A) according to design gained primer annealing temperature, the touchdownPCR of different range is set, to improve amplification efficiency.Such as, certain object region SNP site primer annealing temperature approximate range at 60-55 DEG C, then first with each cycle down 1 DEG C, can arrange the annealing region of 60-55 DEG C.If amplification rear electrophoresis band shows Unspccific bands, or through step b) in qPCR verify that amplification efficiency is not good, in such cases, each cycle down 0.5 DEG C can be selected, 60-55 DEG C is set; Or each cycle down 1 DEG C, arranges 60-50 DEG C;
B) qPCR checking being carried out to multiplex amplification product, detecting the amplification efficiency of each SNP site primer in multiplex amplification, for repeatedly verifying that the primer that amplification efficiency is lower is replaced;
C) arrange different gradient template amount (in such as 50 microlitre systems, select 40,60,80,100,120, the template gradient of 150ng) increase, find out the minimum template amount that amplification efficiency is high, realize multiple SNP amplifications of low template amount;
D) based on regular-PCR amplification, carry out amplification system optimization, improve amplification efficiency.Such as, during amplification, need not common archaeal dna polymerase, and adopt warm start archaeal dna polymerase or high-fidelity DNA polymerase; DNTP and Mg
2+relative proportion is optimized; For the region that GC content is higher, add some toughener etc.
After preliminary experiment success, carry out the amplification of family and embryo's sample.Purifying, Jian Ku, the order-checking of upper machine are carried out to amplified production, according to sequencing data analytical results, carries out SNP-haplotyping.
4. high-flux sequence
Adopt high-flux sequence platform, sample is checked order.Order-checking platform is not particularly limited, s-generation order-checking platform: GA, GAII, GAIIx, HiSeq1000/2000/2500/3000/4000, XTen, XFive, NextSeq500/550, MiSeq of including but not limited to Illumina company, the SOLiD of AppliedBiosystems, IonTorrent, IonPGM, IonProtonI/II of the 454FLX of Roche, ThermoFisherScientific (LifeTechnologies); Third generation single-molecule sequencing platform: the HeliScope system including but not limited to HelicosBioSciences company, the SMRT system of PacificBioscience, GridION, MinION of OxfordNanoporeTechnologies.Order-checking type can be single-ended (SingleEnd) order-checking or both-end (PairedEnd) order-checking.
5. data analysis
5.1 reference sequences comparisons
Sequencing result is removed joint and low quality data, comparison is to reference genome.Full-length genome, arbitrarily karyomit(e), a chromosomal part, gene is can be with reference to genome.Usually select to be recognized the sequence determined with reference to genome, the gene order as NCBI or UCSC is reference sequences or any item chromosome.Comparison software can by any one free or business software, as BWA (Burrows-WheelerAlignmenttool), SOAPaligner/soap2 (ShortOligonucleotideAnalysisPackage), Bowtie/Bowtie2.
5.2SNPcalling
Obtain the SNP of target area with software, software includes but not limited to samtoolsmpileup, in GATK, FreeBayes, VarScan one of at least.
5.3SNP filter
SNP filters, and the order-checking degree of depth is minimum 100 ×, allelotrope heterozygosity difference minimum 10%, the SNP of removing latent fault.
5.4 screening area somatotype SNP, and build male parent and maternal SNP-haplotype.
Differentiation type SNP, i.e. same SNP site, in 4 allelotrope bases of male parent and female parent, having 1 base to be different from other 3 can distinguish.
5.5 analyze embryo SNP-haplotype
Embryo SNP-haplotype has 2, and heredity is from male parent and each 1 of female parent respectively, and according to differentiation type SNP and Mendelian inheritance principle, judge that specifically which is the heredity of father source to its SNP-haplotype, which is source of parents heredity.In embryo SNP-haplotyping, differentiation type SNP is minimum is 10, if there are more than 3 SNP mistakes, this embryo SNP-haplotyping data deficiencies, is difficult to analyze.
5.6 interpretation of result
Determine male parent and maternal SNP-haplotype according to 5.4, distinguish the haplotype chain with pathogenic sites, by the concrete allelotrope contrast of embryo place SNP.According to Mendelian inheritance principle, judge whether embryo carries Disease-causing gene site.
The present invention, by the SNP-haplotyping of aforesaid method for same gene, only needs a set of SNP to detect, without the need to designing different schemes because object is different, shortening the work period, reducing costs.Described in specific as follows:
(1) the present invention adopts multiple PCR technique to catch SNP information, is not limited to, by SNP all in same goal gene target area multiplex amplification simultaneously, also comprise and be divided into several multiplex PCR to catch a whole set of SNP.
(2) in multiplex PCR of the present invention, the primer is not limited to certain a pair or several to primer amplification, every all alternative use of primer for SNP amplification in target area.
(3) the present invention is applied to PGD detection, and be not limited to embryo's full-length genome sample that MALBAC technology obtains, other are applied to the method for a small amount of cell amplification full-length genome, such as, and the methods such as MDA, OmniPlexWGA.
(4) multiple PCR technique of the present invention is not limited to catch a certain specific species SNP information, and can be human genome SNP, also can be other species.
The method utilizing multiple PCR technique to carry out SNP-haplotyping provided by the present invention is applied to PGD and detects, family and embryonic gene group SNP information are caught, embryo SNP information can be determined effectively, accurately, and carry out SNP-haplotyping, determine whether embryo carries genetic mutational site, thus for monogenic disease before Embryonic limb bud cell detect, pregnant woman's antenatal diagnosis or clinical treatment provide foundation.In addition, the present invention also can carry out HLA somatotype, aneuploid detects, and realizes the multinomial detection of single sample.
Embodiment
Below in conjunction with specific embodiment, the present invention is more comprehensively described, exemplary embodiment of the present invention is wherein described.Exemplary embodiment of the present invention and explanation thereof for explaining the present invention, but do not form inappropriate limitation of the present invention.
Embodiment 1
Carry out SNP-haplotyping according to above-mentioned steps, concrete outcome is see table 1.One autosomal recessive hereditary diseases model, Disease-causing gene is A.In this family, male parent and female parent are carrier, carry Mutation1 and Mutation2 mutational site respectively.
By analysis, for A-SNP1, paternal allele type is G/G, maternal allelotype is G/A, and diseased individuals allelotype is G/A, illustrates that maternal this site base A and pathogenic sites interlock in same haplotype, and entailing diseased individuals, this SNP site is differentiation type SNP; All there is base A in the allelotrope of embryo 2,4 and 6, illustrate that these 3 embryos all carry the Disease-causing gene site of source of parents.According to Mendelian inheritance principle, judge whether embryo carries Disease-causing gene site: embryo 1,3 and 5 is father source carriers of mutation, and embryo 2 and 6 is source of parents carriers of mutation, embryo 4 is father and mother source carriers of mutation, i.e. pathogenic embryo.Other sites by that analogy.
Embodiment 2
Carry out SNP-haplotyping according to above-mentioned steps, concrete outcome is see table 2.One X-sex-linked recessive inheritance disease model, Disease-causing gene is B.In this family, female parent is 1 B gene Mutation3 mutational site carrier.Known by as above carrying out analysis, embryo 2 does not carry pathogenic mutation, and embryo 1 and 3 is pathogenic mutation carrier.
Table 1A gene region part SNP genotype
Table 2B gene region part SNP genotype
Annotation:
1. table in " _ " represent cannot obtain corresponding SNP data (without data cover or the degree of depth lower);
2. bold Italic represents pathogenic mutation;
3. in table 1, male parent and maternal haplotype 1 represent pathogenic mutation place haplotype, and in table 2, maternal haplotype 1 represents pathogenic mutation place haplotype;
4., in table 1 and table 2, Mutation1, Mutation2 and Mutation3 are respectively pathogenic mutation.
Although explained specific embodiment of the present invention by example, it should be appreciated by those skilled in the art, above example is only to be described, instead of in order to limit the scope of the invention.It should be appreciated by those skilled in the art, can without departing from the scope and spirit of the present invention, above embodiment be modified.Scope of the present invention is limited to the appended claims.
In sum, invention applies multiple PCR technique and catch SNP target area, in conjunction with high-flux sequence, carry out SNP-haplotyping, in the past conventional STR genetic marker can be solved few, by the genetic marker distance objective region problem easily producing restructuring comparatively far away, tens of even hundreds of SNP site are carried out Single tube amplification, simple to operate, work period is short, cost is lower, especially in conjunction with high-flux sequence, detect and data handling procedure very quick.In addition, this invention also solves the full-length genome sample obtained by a small amount of cell in experimentation, easily occur this series of problem of the phenomenon such as ADO, pollution, whether embryo's sample carries pathogenic mutation site to utilize SNP-haplotyping to determine.Meanwhile, the present invention, in conjunction with MALBAC technology, can carry out the SNP-haplotyping of trace sample.
Claims (10)
1. utilize multiple PCR technique to carry out a method for SNP-haplotyping, it is characterized in that, comprise the steps:
(1) according to target area design primer
(1.1) target gene is determined, according to concrete kind, with NCBI official website gene order for the particular location with reference to gene determination target gene designation of chromosome, thus lock acquisition region;
(1.2) SNP region is selected, be defined within the scope of target gene upstream and downstream 1M, determine SNP areas captured scope further, catching clip size at SNP site upstream and downstream 100bp is about 200bp, primer-design software or primer Specialty Design website is utilized to carry out design of primers, after design of primers completes, specificity assessment is carried out to primer, assess qualified after carry out primer synthesis;
(2) preparation of family sample and embryo's sample
(2.1) extract diseased individuals in family, male parent, maternal peripheral blood sample, obtain genomic dna;
(2.2) embryo collection cell sample, carries out whole genome amplification;
(3) multiplexed PCR amplification, carries out catching of target area SNP to family and embryo's sample;
(4) adopt high-flux sequence platform, sample is checked order;
(5) data analysis
(5.1) sequencing result that step (4) obtains is removed joint and low quality data, adopt the comparison of comparison software to reference genome;
(5.2) obtain the SNP of target area with software, described software includes but not limited to samtoolsmpileup, at least one in GATK, FreeBayes, VarScan;
(5.3) SNP filters: the degree of depth that checks order is minimum is 100 × and, allelotrope heterozygosity difference is minimum is 10%, the SNP of removing latent fault;
(5.4) screening area somatotype SNP, and build male parent and maternal SNP-haplotype: same SNP site, in male parent and 4 maternal allelotrope bases, having 1 base to be different from other 3 can distinguish;
(5.5) analyze SNP-haplotype: embryo SNP-haplotype has 2, heredity is from male parent and each 1 of female parent respectively, and according to differentiation type SNP and Mendelian inheritance principle, judge that specifically which is the heredity of father source to its SNP-haplotype, which is source of parents heredity;
(5.6) interpretation of result: determine male parent and maternal SNP-haplotype according to step (5.4), distinguish the haplotype chain with pathogenic sites, by the concrete allelotrope contrast of embryo place SNP, according to Mendelian inheritance principle, judge whether embryo carries Disease-causing gene site.
2. method according to claim 1, is characterized in that, the design of primers described in step (1.2), the annealing temperature of each SNP site primer is controlled, in less scope, to be beneficial to follow-up pcr amplification during design.
3. method according to claim 2, is characterized in that, the top temperature of described annealing region and the difference of minimum temperature are not more than 10 DEG C.
4. method according to claim 1, is characterized in that, primer length designed in step (1.2) is 20-25bp.
5. method according to claim 1, is characterized in that, all primers equimolar ratio example when synthesizing of design in step (1.2) merges into a pipe, when follow-up multiplexed PCR amplification as single primer amplification.
6. method according to any one of claim 1 to 5, is characterized in that, adopts MALBAC technology to complete whole genome amplification in step (2.2).
7. method according to any one of claim 1 to 5, is characterized in that, when carrying out multiplexed PCR amplification in step (3), adopts touchdownPCR amplification program, covers multipair primer annealing temperature, finally reach the coverage of 90%.
8. method according to claim 7, is characterized in that, step (3) adopts warm start archaeal dna polymerase or high-fidelity DNA polymerase when increasing.
9. method according to claim 8, is characterized in that, the order-checking type in step (4) is single-ended order-checking or both-end order-checking.
10. method according to claim 9, is characterized in that, in the embryo SNP-haplotyping of step (5.5), differentiation type SNP is minimum is 10, if there are more than 3 SNP mistakes, this embryo SNP-haplotype data is considered as data volume deficiency, is difficult to analyze.
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| CN114728069A (en) * | 2019-09-30 | 2022-07-08 | 迈欧米公司 | Multi-gene risk score for in vitro fertilization |
| CN114836536A (en) * | 2022-07-04 | 2022-08-02 | 北京大学第三医院（北京大学第三临床医学院） | Method and system for screening single-cell high-amplification region based on MALBAC |
| CN116855617A (en) * | 2023-08-31 | 2023-10-10 | 安诺优达基因科技(北京)有限公司 | Series repeated variation typing detection method based on core family and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519689A (en) * | 2008-12-10 | 2009-09-02 | 复旦大学附属中山医院 | Construction and application of polymorphism and haplotype of tumor metastasis related gene osteopontin |
| CN102121046A (en) * | 2009-06-25 | 2011-07-13 | 中国科学院北京基因组研究所 | Chinese population linkage analysis single nucleotide polymorphism (SNP) marker sets and use method and application thereof |
| CN103492588A (en) * | 2011-02-25 | 2014-01-01 | 伊路敏纳公司 | Methods and systems for haplotype determination |
| CN104004817A (en) * | 2013-02-22 | 2014-08-27 | 哈佛大学 | Selection of embryo of test tube baby through sequencing by single cell genome of polar body or embryo |
| CN104862380A (en) * | 2014-02-25 | 2015-08-26 | 林巍 | Familial specific genetic disease correlated allele haplotype variation tag confirmation method |
-
2015
- 2015-11-05 CN CN201510749459.6A patent/CN105385755A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101519689A (en) * | 2008-12-10 | 2009-09-02 | 复旦大学附属中山医院 | Construction and application of polymorphism and haplotype of tumor metastasis related gene osteopontin |
| CN102121046A (en) * | 2009-06-25 | 2011-07-13 | 中国科学院北京基因组研究所 | Chinese population linkage analysis single nucleotide polymorphism (SNP) marker sets and use method and application thereof |
| CN103492588A (en) * | 2011-02-25 | 2014-01-01 | 伊路敏纳公司 | Methods and systems for haplotype determination |
| CN104004817A (en) * | 2013-02-22 | 2014-08-27 | 哈佛大学 | Selection of embryo of test tube baby through sequencing by single cell genome of polar body or embryo |
| CN104862380A (en) * | 2014-02-25 | 2015-08-26 | 林巍 | Familial specific genetic disease correlated allele haplotype variation tag confirmation method |
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| WO2021018197A1 (en) * | 2019-07-30 | 2021-02-04 | 苏州亿康医学检验有限公司 | Analysis method for determining haplotypes of filial generation objects and device |
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| CN113005195B (en) * | 2021-04-27 | 2021-12-14 | 北京嘉宝仁和医疗科技有限公司 | Primer composition, product and method for detecting embryo carrying risk chromosome |
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