CN110305968A - A kind of composite amplification system in the micro- haplotype domain SNP-DIP based on NGS parting for medical jurisprudence individual identification - Google Patents
A kind of composite amplification system in the micro- haplotype domain SNP-DIP based on NGS parting for medical jurisprudence individual identification Download PDFInfo
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Abstract
The invention belongs to medical jurisprudence new technology innovation research application fields, and in particular to disclose a kind of compound amplification detection system in micro- haplotype domain that the SNP-DIP molecular genetic marker for medical jurisprudence individual identification forms.The present invention is based on the sites SNP or DIP of the past report, using the research strategy in micro- haplotype domain, the flanking sequence (each 100bp up and down) in these sites is consulted with the presence or absence of other SNP or DIP hereditary variations by the library dbSNP, according to the composite type difference of hereditary variation in each region, the molecular genetic marker for being similar to micro- haplotype domain is formed.The present invention carries out deep sequencing to these regions, it can be achieved that the synchronization parting of multiple samples detects, and can get all sites SNP or DIP base sequence variation information in each region using the method for two generations sequencing.18 micro- haplotype domains cumulative individual discrimination with higher in the group of East Asia provided by the invention, can be used for the application study of medical jurisprudence individual identification.
Description
Technical field
The invention belongs to medical jurisprudence new industrial research development fields, and in particular to disclose a kind of single nucleotide polymorphism and lack
The composite amplification system in the micro- haplotype domain of mistake/insertion polymorphic molecular genetic marker composition and its analysis being sequenced based on two generations
Detection method.
Background technique
Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is single nucleosides in genome
Acid variation is formed by sequence polymorphism molecular genetic marker；Missing/insertion polymorphism (Deletion/Insertion
Polymorphism, DIP) it is that the DNA fragmentation of insertion or missing different length is formed by length polymorphism molecule in genome
Genetic marker；Both genetic markers are low with widely distributed in genome, mutation rate and easily obtain the excellent of shorter amplicon
Gesture.SNP and DIP genetic marker is mainly shown as the hereditary variation of two allele, compares str locus seat, both heredity marks
The genetic polymorphism of note is relatively relatively low.Therefore in forensic application, more sites SNP and DIP are needed to can be only achieved existing
Str locus seat medical jurisprudence efficiency obtained, realize medical jurisprudence individual identification and paternity identification purpose.
As a kind of novel molecular genetic marker, it is by (small in one section in genome specific region in micro- haplotype domain
In 200bp) multiple sites SNP and/or DIP composed by genetic marker, the combination of these SNP and/or DIP constitute micro-
The diversity of haplotype.Therefore micro- haplotype domain genetic marker compares SNP or DIP genetic marker, has higher polymorphism, together
When have both other advantages of SNP or DIP genetic marker, there is huge application potential in medical jurisprudence research.
Two generation sequencing technologies (Next Generation Sequencing, NGS), also known as large-scale parallel sequencing, are compared
Generation sequencing technologies are not limited by fluorescent material type and quantity, can not only detect multiple samples simultaneously, but also can be real
The now Synchronization Analysis detection of a large amount of genetic markers；Sequence polymorphism analysis can be carried out while analysis length polymorphism, so
NGS technology has certain advantage for analysis micro- haplotype domain genetic marker.
The present invention uses the research strategy in micro- haplotype domain, based on SNP the or DIP genetic marker of the past report, passes through
Its upstream and downstream (in each 100bp) of dbSNP library inquiry whether there is other hereditary variations (SNP or DIP), filter out those flank sequences
There is SNP the or DIP genetic marker of other variations in column, then use two generation platform sequencing analysis these genetic markers.
Summary of the invention
The object of the present invention is to provide a kind of composite amplification system in micro- haplotype domain of SNP-DIP composition and its it is based on
NGS genotyping detection method.In order to realize the present invention, we use following technical scheme:
1, there is the screening of the SNP/DIP genetic marker of variation in flanking sequence.
Firstly, we based on previously studying the site SNP or DIP reported, pass through these sites of dbSNP library lookup
With the presence or absence of other variations in the region of each 100bp of upstream and downstream, the screening principle in site is as follows: (1) existing in each region
The minimum gene frequency in the site SNP/DIP is greater than 0.01；(2) site SNP/DIP in each region is not at strong chain
Non-equilibrium state；(3) physical distance of different zones is greater than 10Mb or in different chromosomes；(4) each region and it is existing often
The physical distance of STR genetic marker is greater than 10Mb or in different chromosomes；(5) site SNP/DIP in each region
The haplotype constituted meets hardy weinberg equilibrium in the group of East Asia.
Based on the above principle, 18 regions are selected in this research altogether, and the rs in each included site in region is numbered, place
Chromosome and specific position are as shown in table 1.The name in site is named based on the principle of the past report in each region,
By taking MH01ZBF002 as an example, MH indicates the english abbreviation of micro- haplotype (Microhaplotype), and 01 indicates No. 1 chromosome, ZBF
Indicate the laboratory in this site of discovery, 002 for further discriminating between the micro- haplotype domains of difference found on same chromosome
Site.
The site SNP/DIP and its essential information in each region of the screening of table 1.
2, the building of the site composite amplification system screened.The present invention is using 3 online tool of Primer to each of preliminary screening
Region carries out design of primers, then using the primer efficiency of Oligo software entry evaluation design, and carries out to each pair of primer single
PCR amplification, agarose gel electrophoresis detection further assessment primer efficiency.After the primer sequence for determining each region,
The present invention realizes the synchronous amplification analysis in 18 regions by adjusting the ratio of different zones inner primer concentration.18 regions
The reaction condition of multiplex PCR is as follows: carrying out first round PCR to 18 regions first, PCR system is 10 μ L, specifically includes 3.1 μ L
ddH2O、1µL 10×PCR buffer、2µL 50nM Primer Mix、0.8µL 2.5mM dNTP、0.1µL 5U/µL Hot
Start DNA polymerase, 1 μ L 100mM Mg2+With 2 μ L sample DNAs.PCR reaction condition are as follows: 95 DEG C of denaturation 15min；94℃
It is denaturalized 30s, 60 DEG C of annealing 10min, 72 DEG C of extension 30s, totally 4 circulations；Then, 94 DEG C of denaturation 30s, 60 DEG C of annealing 1min, 72
DEG C extend 30s, totally 15 circulation.
Then the second wheel PCR is carried out again.Before carrying out PCR, need that 100 μ L ddH are added to first round PCR product2O
It is diluted.It includes 3.5 μ L ddH that PCR product after taking 10 μ L to dilute, which is added to,2O、1µL 10×PCR buffer、3.6µ
2 μM of barcode of L, 0.8 μ L 2.5mM dNTP, 0.1 μ L 5U/ μ L Hot Start DNA polymerase and 1 μ L 100mM
Mg2+Mixed solution in.PCR reaction condition is as follows: 95 DEG C of denaturation 15min；94 DEG C of denaturation 30s, 60 DEG C of annealing 4min, 72 DEG C are prolonged
30s is stretched, totally 5 circulations；Then 94 DEG C of denaturation 30s, 65 DEG C of annealing 1min, 72 DEG C of extension 30s, totally 10 circulations, 4 DEG C of preservations are standby
With.
3, the purifying in library and quantitative.The second wheel PCR product is taken, agarose gel electrophoresis is carried out, then to target fragment
Gel extraction is carried out, purification process is carried out to target fragment using Tiangeng DP214 kit.Then raw using Agilent 2100
Object analyzer quantifies library.
4, machine sequencing and data analysis on library.It is flat that the library that step 3 is built is placed in illumina X-10 sequencing
On platform, sequencing analysis is carried out.For the sequencing data of acquisition, joint sequence is removed using Cutadapt software first, is then adopted
The sequence alignment after removing connector to the mankind is referred on genome (GRCh38.p12) with BWA software, it is true by GATK software
Surely the Genotyping in site is studied.
The present invention provides one kind can detect the site SNP/DIP and the parting detection architecture of its flanking sequence simultaneously, specifically
Site information including 18 regions, system component, the reaction condition of two step PCR, library is quantitative, upper machine sequencing and subsequent number
According to operating procedures such as analyses.The present invention is based on the Research Thinkings in micro- haplotype domain to be led to using the site SNP/DIP of the past report
It crosses the library dbSNP and consults the upstream and downstream in these sites with the presence or absence of other hereditary variations, according to the hereditary variation group of its flanking sequence
At the genetic marker for being similar to micro- haplotype domain, genetic polymorphism and the legal medical expert in the single site SNP/DIP can further improve
Learn effectiveness；The present invention using two generations sequencing method these regions are tested and analyzed, it can be achieved that multiple samples it is same
Step tests and analyzes, and can get all hereditary variation information in each region；The amplification in each region that the present invention screens
Son is in 300bp hereinafter, the analysis detection for the sample that can be used for degrading.
Specific embodiment
Below by taking a sample as an example, the operating process that present invention be described in more detail.It should be pointed out that following say
Bright is only to claimed technical solution for example, not to any restrictions of these technical solutions.This
The protection scope of invention be subject to the appended claims record content.
Reagent used in the present invention: ddH2O、10×PCR buffer、50nM Primer Mix、2.5mM dNTP、
5U/ μ L Hot Start DNA polymerase, 100mM Mg2+With 2 μM of barcode reagents.
Detection method.
(1) the 2 μ L of DNA for taking individual sample, the PCR reaction system provided using the present invention are as follows.
| Component | Volume |
| ddH2O | 3.1µL |
| 10×PCR buffer | 1µL |
| 50nM Primer Mix | 2µL |
| 2.5mM dNTP | 0.8µL |
| 5U/ μ L Hot Start DNA polymerase | 0.1µL |
| 100mM Mg2+ | 1µL |
| Sample DNA | 2µL |
PCR condition is as follows.
After completing first round PCR reaction, processing is diluted to PCR product.Take 100 μ L ddH2O is added to the first round
PCR reagent in, concussion mix after, take the sample of 10 μ L by following system carry out second wheel PCR.
| Component | Volume |
| ddH2O | 3.5µL |
| 10×PCR buffer | 1µL |
| 2µM barcode | 3.6µL |
| 2.5mM dNTP | 0.8µL |
| 5U/ μ L Hot Start DNA polymerase | 0.1µL |
| 100mM Mg2+ | 1µL |
| PCR product after dilution | 10µL |
PCR condition is as follows.
(2) purifying in library and quantitative: after completing two-step pcr, the second wheel PCR product is taken, it is electric to carry out Ago-Gel
Then swimming detection is chosen target fragment and carries out gel extraction, carried out at purifying using Tiangeng DP214 kit to target fragment
Reason.The concentration in library is finally determined using 2100 biological analyser of Agilent.
(3) sequencing point machine sequencing and data analysis on: is carried out to the library built using illumina X-10 sequenator
Analysis.The data of acquisition use Cutadapt software to remove joint sequence first, then use BWA software by the sequence after removing connector
Column and the mankind are compared with reference to genome (GRCh38.p12), and the Genotyping in research site is determined by GATK software.
Claims (4)
1. a kind of compound amplification detection system in micro- haplotype domain that the SNP-DIP for medical jurisprudence individual identification is formed, special
Sign is the site information in the micro- haplotype region 18 SNP/DIP；The system proportion and reaction condition of 18 region two-step pcrs.
2. the site information in 18 regions for including in the system is as follows according to claims 1.
3. component included in the reagent of research and development has ddH according to claims 12O、10×PCR buffer、50nM
Primer Mix, 2.5mM dNTP, 5U/ μ L Hot Start archaeal dna polymerase, 100mM Mg2+With 2 μM of barcode reagents.
4. according to claim 1, the operating procedure of the system of research and development is as follows: (1) sample to be tested is taken, using magnetic bead
DNA extraction kit extracts sample DNA；(2) carrying out first round PCR:PCR system first is 10 μ L, is specifically included: 3.1 μ L
ddH2O、1µL 10×PCR buffer、2µL 50nM Primer Mix、0.8µL 2.5mM dNTP、0.1µL 5U/µL Hot
Start DNA polymerase, 1 μ L 100mM Mg2+With 2 μ L sample DNAs；PCR reaction condition are as follows: 95 DEG C of denaturation 15min；94℃
It is denaturalized 30s, 60 DEG C of annealing 10min, 72 DEG C of extension 30s, totally 4 circulations；Then 94 DEG C of denaturation 30s, 60 DEG C of annealing 1min, 72 DEG C
Extend 30s, totally 15 circulations；(3) the second wheel PCR is then carried out again: before carrying out PCR, needing the PCR product to the first round
100 μ L ddH are added2O is diluted；It includes 3.5 μ L ddH that PCR product after taking 10 μ L to dilute, which is added to,2O、1µL 10×
PCR buffer, 3.6 μ L, 2 μM of barcode, 0.8 μ L 2.5mM dNTP, 0.1 μ L 5U/ μ L Hot Start DNA polymerization
Enzyme and 1 μ L 100mM Mg2+Mixed solution in；PCR reaction condition is as follows: 95 DEG C of denaturation 15min；94 DEG C of denaturation 30s, 60 DEG C
Anneal 4min, 72 DEG C of extension 30s, totally 5 circulations；Then 94 DEG C of denaturation 30s, 65 DEG C of annealing 1min, 72 DEG C of extension 30s, totally 10
A circulation；(3) purifying in library and quantitative: the second wheel PCR product is taken, agarose gel electrophoresis detection is carried out, then to purpose
Segment carries out gel extraction, carries out purification process to target fragment using Tiangeng DP214 kit；Then Agilent is used
2100 biological analysers quantify library；(4) machine sequencing and data analysis on: the library built is placed in
Sequencing analysis is carried out in illumina X-10 microarray dataset；For the sequencing data of acquisition, gone first using Cutadapt software
Except joint sequence, then the sequence after removing connector and the mankind are carried out with reference to genome (GRCh38.p12) using BWA software
It compares, the Genotyping in research site is determined by GATK software.
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| CN111518917A (en) * | 2020-04-02 | 2020-08-11 | 中山大学 | Micro haplotype genetic marker combination and method for non-invasive prenatal paternity determination |
| CN112342303A (en) * | 2020-12-04 | 2021-02-09 | 郑州高新生物技术有限公司 | NGS-based human Y chromosome STR and SNP genetic marker combined detection system and detection method |
| CN112885407A (en) * | 2021-01-29 | 2021-06-01 | 杭州联川基因诊断技术有限公司 | Second-generation sequencing-based micro-haplotype detection and typing system and method |
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| CN111518917A (en) * | 2020-04-02 | 2020-08-11 | 中山大学 | Micro haplotype genetic marker combination and method for non-invasive prenatal paternity determination |
| CN111518917B (en) * | 2020-04-02 | 2022-06-07 | 中山大学 | Micro haplotype genetic marker combination and method for noninvasive prenatal paternity relationship determination |
| CN112342303A (en) * | 2020-12-04 | 2021-02-09 | 郑州高新生物技术有限公司 | NGS-based human Y chromosome STR and SNP genetic marker combined detection system and detection method |
| CN112885407A (en) * | 2021-01-29 | 2021-06-01 | 杭州联川基因诊断技术有限公司 | Second-generation sequencing-based micro-haplotype detection and typing system and method |
| CN114634988A (en) * | 2022-04-28 | 2022-06-17 | 贵州医科大学 | SNP (Single nucleotide polymorphism) sites and method for identifying and researching biological geographic sources of east Asia population |
| CN114634988B (en) * | 2022-04-28 | 2022-09-16 | 贵州医科大学 | SNP (Single nucleotide polymorphism) sites and method for identifying and researching biological geographic sources of east Asia population |
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