CN110499372A - Multiplex PCR targeted capture parting system and kit based on high throughput sequencing technologies - Google Patents
Multiplex PCR targeted capture parting system and kit based on high throughput sequencing technologies Download PDFInfo
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Abstract
The present invention discloses a kind of multiplex PCR targeted capture parting system and kit based on high throughput sequencing technologies, is related to nucleic acid in vitro detection technique field.The STR classification system includes the PCR primer for expanding 58,119 or 179 chain euchromosome STR locus, which includes PCR primer combination, Index joint sequence, IGT-EM707 polymerase mixture, amplification buffering reinforcing agent NB, YF buffer solution B and build library reagent etc..STR classification system and its kit provided by the invention can be realized the Single tube amplification to 58,119 or 179 chain euchromosome STR locus, balance is good, high sensitivity, specificity is good, genotyping result is accurate, can be used for identifying the complicated affiliation of same level-one difference affiliation.
Description
Technical field
The present invention relates to legal medical expert's detection technique fields, and in particular to a kind of multiplex PCR target based on high throughput sequencing technologies
To the compound parting system and its kit of capture sequencing.
Background technique
Short tandem repeatSTR (short tandem repeat, STR) is the special sequence being composed in series by 2-6bp recurring unit
Column, str locus seat quantity is big, widely distributed, accounts for about the 3% of whole gene group, and polymorphism is high, polymorphism is derived mainly from core
The difference of heart sequence repetition number between individuals, this species diversity follow mendelian inheritance in genetic process.Therefore, STR
Augmentation detection technology is widely used in medical jurisprudence individual identification, relationship identification and population genetic study.Most common STR parting
Technology is the typing method of fluorescent marker multiplex amplification combination Capillary Electrophoresis (capillary electrophoresis, CE)
With two generation sequencing technologies.
The identification of complicated affiliation is current judicial expertise field one of technical problem in the urgent need to address, in the administration of justice
It is chiefly used in lawsuit, inheritance in practice, remains claims, traffic accident, migrates case and find relatives in large-scale disaster
Etc. multiple fields, such case has the tendency that increasing in recent years.Therefore the success or not of this kind of case identification often depends on
The genetic connection and social relationships of several families are not only huge challenge but also to the personal of common people to medicolegal genetics man
Interests have significant impact.
Can face half sibs and uncle (aunt) nephew in complicated Relationship iden- tification, half sibs and grandfather (milk) grandson or uncle and nephew with
The demand identified between different affiliations in this kind of same level-one between grandfather grandson, but these three types of relationships belong to second level, pass through Meng
Identity by decent (IBD) value and coefficient of coancestry (θ) value that Dare law of inheritance is derived all are consistent, so answering
These three relationships can not be distinguished with existing common independent inheritance label and calculation method.There is scholar's proposition, though
The identification of right this kind of same first degree relative can pass through age information and other DNA informations, such as mitochondrial DNA, property dyeing
Body DNA can assist the judgement of problems, but in face of the ordinary circumstance with level-one difference affiliation, chain autosome
More than genetic marker can be solved the problems, such as preferably.In theoretical research, Thompson uses linkage inheritance law within 1998
Recombination fraction be deduced grandfather grandson, half sibs, affiliation coefficient different between uncle and nephew three, as computer technology is in method
The application in material evidence field is cured, being made that in Egeland in 2008 according to the different affiliation coefficients that Thompson is obtained makes
Theoretical calculation model is made that with a certain number of linkage inheritances label.
At present about the prior art of Relationship iden- tification, such as: Chinese patent CN104818323B disclose the mankind 13,
The gene parting detecting reagents of 18 and No. 21 chromosome, 20 str locus seats is, it can be achieved that single tube to 20 str locus seats
Amplification；Chinese patent CN106906292A discloses 22 short tandem repeat composite amplification methods of one kind and its kit,
The kit can be used for expanding 22 str locus seats and 1 gender locus.Most of chain STR research is all X-STR and Y-
STR genetic marker, such genetic marker need special affiliation that could apply；The euchromosome STR of independent inheritance is not yet
Complicated affiliation can be distinguished.
The present invention is directed to the characteristics of chain euchromosome STR, customizes the STR classification system of multiplex PCR targeted capture sequencing,
STR classification system can disposably amplify all STR genetic markers, and used primer composite sequence balance is good, energy
All locus are detected in enough guarantee groups, are analyzed by the sequencing of two generations and subsequent data, available each STR's
Parting and sequence information can be used for identifying the complicated affiliation of same level-one difference affiliation.
Summary of the invention
The purpose of the present invention is to provide a kind of STR classification systems and its kit for complicated Relationship iden- tification.
The technical problems existing in the prior art are overcome, the STR classification system and its kit can be realized to 179 chain normal dyes
The Single tube amplification of colour solid str locus seat, balance is good, high sensitivity, specificity is good, genotyping result is accurate, can be used for identifying same
The complicated affiliation of level-one difference affiliation.
In another aspect, finding in research process, above-mentioned 179 str locus groups can be divided into three groups independent of each other, this three
Group str locus seat is independent or the combination of 6 kinds of str locus seats of composition is cooperated to can be realized and above-mentioned 179 str locus seats two-by-two
Identical, the similar or equivalent technical effect of the classification system of composition.
More than being based on:
The first, the present invention provides a kind of STR classification system for complicated Relationship iden- tification, the STR partings
System includes the PCR primer combination 1 for expanding 60 chain euchromosome STR locus；
The chain euchromosome STR locus of described 60, corresponding physical location, dyeing on reference genome Hg38
Body subregion and genetic distance are as follows:
The PCR primer combination 1 includes forward primer group and reverse primer group；
The forward primer group are as follows:
The reverse primer group are as follows:
The second, the present invention provides a kind of STR classification system for complicated Relationship iden- tification, the STR partings
System includes the PCR primer combination 2 for expanding 58 chain euchromosome STR locus；
The chain euchromosome STR locus of described 58, corresponding physical location, dyeing on reference genome Hg38
Body subregion and genetic distance are as follows:
The PCR primer combination 2 includes forward primer group and reverse primer group；
The forward primer group are as follows:
The reverse primer group are as follows:
Third, the present invention provides a kind of STR classification system for complicated Relationship iden- tification, the STR partings
System includes the PCR primer combination 3 for expanding 61 chain euchromosome STR locus；
The chain euchromosome STR locus of described 61, corresponding physical location, dyeing on reference genome Hg38
Body subregion and genetic distance are as follows:
The PCR primer combination 3 includes forward primer group and reverse primer group；
The forward primer group are as follows:
The reverse primer group are as follows:
4th, the present invention provides a kind of STR classification system for complicated Relationship iden- tification, the STR partings
System includes the PCR primer combination 4 for expanding 118 chain euchromosome STR locus；The chain normal dye of described 118
Colour solid str locus seat is the str locus seat of STR bit point serial number 1-118 during description of the invention is recorded；The PCR primer
Combination 4 is drawn for the forward and reverse for expanding the str locus seat of STR bit point serial number 1-118 in description of the invention record
Object combination, specifically combines 1 and 2 by primer and forms.
5th, the present invention provides a kind of STR classification system for complicated Relationship iden- tification, the STR partings
System includes the PCR primer combination 5 for expanding 121 chain euchromosome STR locus；The chain normal dye of described 121
Colour solid str locus seat is the str locus seat of STR bit point serial number 1-60 and 119-179 during description of the invention is recorded；Described
PCR primer combination 5 is the str locus seat for expanding STR bit point serial number 1-60 and 119-179 in description of the invention record
Forward and reverse primer combination, specifically combine 1 and 3 by primer and form.
6th, the present invention provides a kind of STR classification system for complicated Relationship iden- tification, the STR partings
System includes the PCR primer combination 6 for expanding 119 chain euchromosome STR locus；The chain normal dye of described 119
Colour solid str locus seat is the str locus seat of STR bit point serial number 61-179 during description of the invention is recorded；The PCR primer
Combination 6 is drawn for the forward and reverse for expanding the str locus seat of STR bit point serial number 61-179 in description of the invention record
Object combination, specifically combines 2 and 3 by primer and forms.
7th, the present invention provides a kind of STR classification system for complicated Relationship iden- tification, the STR partings
System includes the PCR primer combination 7 for expanding 179 chain euchromosome STR locus；The chain normal dye of described 179
Colour solid str locus seat is the str locus seat of STR bit point serial number 1-179 during description of the invention is recorded；The PCR primer
Combination 7 is drawn for the forward and reverse for expanding the str locus seat of STR bit point serial number 1-179 in description of the invention record
Object combination, specifically combines 1,2 and 3 by primer and forms.
For more intuitive embodiment technical solution of the present invention, above the first to seven drip irrigation devices are summarized as follows:
8th, the present invention provides a kind of kit for complicated Relationship iden- tification, the kit includes this
The PCR primer combination 1,2,3,4,5,6 or 7 recorded in description of the invention.
In the primer combination, positive and negative primer concentration is 0.1 μM.
Further, the kit further includes Index joint sequence and archaeal dna polymerase；
It is further preferred that the Index joint sequence includes IGT-I5 Index and IGT-I7 Index, working solution
Concentration is 10 μM.
Index joint sequence, which is that illumina microarray dataset institute is general, obtains joint sequence, as the differentiation between sample.
Preferably, the kit further includes the reagent that genomic DNA is made to the library for sequencing.
Another aspect of the present invention additionally provides a kind of method of complicated Relationship iden- tification, and the method includes following step
It is rapid:
(1) human gene group DNA is extracted, quantitative genomic DNA concentration is 1-20ng/ μ L；
(2) multiplex PCR library construction:
A. through first round multi-PRC reaction, first round magnetic beads for purifying, multiple PCR products after purification are obtained；
B. using multiple PCR products after purification as template, the second wheel multi-PRC reaction is carried out, Index connector sequence is inserted into
Column obtain multiplex PCR library through the second wheel magnetic beads for purifying；
C. quantitative and quality testing is carried out to obtained multiplex PCR library；
(3) the multiplex PCR library obtained to step (2) carries out sequencing and data analysis, obtains the corresponding STR of each individual
Genotyping result and medical jurisprudence parameter.
It is further preferred that genomic DNA concentration is 10-20ng/ μ L.
The STR classification system and kit are suitable for all samples comprising DNA, including but not limited to blood, essence
Spot, hair, bone, skin, solid tissue etc..
Compared with prior art, positive and beneficial effect of the invention is:
(1) a kind of STR classification system and its kit for complicated Relationship iden- tification provided by the present invention, can
60,58,61,118,121,119 or 179 chain euchromosome STR locus are disposably detected simultaneously, are establishing multiplex PCR
During amplification system, with the increase of detected str locus seat number, interfering with each other each between of primer also be will increase, this
Invention realizes the Single tube amplification to the chain euchromosome STR locus of the STR of above-mentioned various quantity by the design of optimization,
STR classification system and its kit balance are good.
(2) in the prior art, the chain STR genetic marker of the euchromosome STR and sex chromosome of independent inheritance, cannot
Identification such as half sibs and uncle (aunt) nephew, this kind of same level-one difference relationship between half sibs and grandfather (milk) grandson or uncle and nephew and grandfather grandson
The complicated affiliation of relationship.And STR classification system provided by the invention and its kit are for chain euchromosome STR
Feature can be used for the complicated affiliation of this kind of identification, and qualification result is accurate.
(3) STR classification system and its kit provided by the invention are reacted using two-wheeled PCR and carry out library construction, had
Many advantages, such as library construction period is short, and comparison rate is high, and capture rate is good, reproducible, easy to operate, testing result it is sensitive
Degree is high, and specificity is good, and genotyping result is accurate, disposably can effectively expand 60,58,61,118,121,119 or 179 it is chain often
Chromosome str locus seat.
It will be clear that the invention discloses 179 str locus seats of documented whole and its accordingly expanding just
To on the basis of primer and reverse primer, those skilled in the art can according to the disclosure of the present invention, rational expectation: adopting
Any matched combined or corresponding positive with Genetic Detection efficiency quantity in 179 STR bit points documented by the present invention
The technical solution constituted with the matched combined of reverse primer, can obtain the technical effect equivalent or similar with the present invention.Institute
Any any integer being specifically as follows between 3-179 with Genetic Detection efficiency quantity stated.Therefore, remembered by this section of content
The technical solution that load mode obtains extends, in protection scope and infringement range in technology of the invention.
Detailed description of the invention
Fig. 1 is the quality inspection peak figure using the library of the combination preparation of primer disclosed in embodiment 1.
Fig. 2 is the quality inspection peak figure using the library of the combination preparation of primer disclosed in embodiment 2.
Fig. 3 is the quality inspection peak figure using the library of the combination preparation of primer disclosed in embodiment 3.
Fig. 4 is the quality inspection peak figure using the library of the combination preparation of primer disclosed in embodiment 4.
Fig. 5 is the quality inspection peak figure using the library of the combination preparation of primer disclosed in embodiment 5.
Fig. 6 is the quality inspection peak figure using the library of the combination preparation of primer disclosed in embodiment 6.
Fig. 7 is the quality inspection peak figure using the library of the combination preparation of primer disclosed in embodiment 7.
Specific embodiment
Below by specific embodiments of the present invention will be described in detail.In order to avoid excessive unnecessary details, In
It will not be described in detail in following embodiment to belonging to well known structure or function.In addition to being defined, institute in following embodiment
Technical and scientific term has the identical meanings being commonly understood by with those skilled in the art of the invention.
A kind of embodiment 1: STR classification system and its kit for complicated Relationship iden- tification
The chain str locus seat of 1.1 autosomes constitutes and characteristic:
(1) the STR classification system in the present embodiment includes following 60 str locus seats:
D1S2131、D1S3721、D1S2130、D1S1600、D1S1653、D1S1660、D1S3732、D2S1364、
D2S2734、D2S1396、D2S428、D2S435、D2S1387、D2S1792、D2S1399、D2S2959、D3S2431、
D3S4547、D4S1643、D4S2408、D4S3326、D4S2368、D5S2845、D5S1473、D5S813、D5S1716、
D5S1459、D5S1487、D5S1466、D5S2496、D5S2501、D6S1019、D6S2417、D6S2412、D6S1284、
D7S1820、D7S3050、D7S821、D8S594、D8S1468、D8S2322、D8S569、D8S1475、D9S746、D9S319、
D9S2149、D11S2010、D11S1392、D12S376、D12S1052、D13S317、D13S790、D14S1432、D14S1428、
D15S644, D15S1507, D16S752, D16S485, D20S481 and D20S1151；
It is with following measurable properties:
1) core space recurring unit is tetranucleotide；
2) heterozygosity of each STR is greater than 0.6；
3) every group of STR number at least two for including and its genetic distance are less than 3cM；
4) each STR has the name of DXSXX.
(2) it is used for while expanding the primer combination 1 of 60 str locus seats in the present embodiment are as follows:
Table 1: the forward primer group of primer combination 1
Table 2: the reverse primer group of primer combination 1
(3) working concentration of forward primer is 0.1 μM in the present embodiment；The working concentration of reverse primer is 0.1 μM.
A kind of 1.2 kits for complicated Relationship iden- tification:
Including above-mentioned PCR primer combination 1, Index joint sequence, IGT-EM707 polymerase mixture.
The Index joint sequence includes IGT-I5 Index and IGT-I7 Index, and working solution concentration is 10 μM.
Index joint sequence are as follows: the I7 of IGT-I7 index Ai Jitaikang terminates header sequence, and working concentration is 10 μM；One
A includes 96, joint sequence information such as following table
The I5 of IGT-I5 Index Ai Jitaikang terminates header sequence, and working concentration is 10 μM；One includes 4, connector sequence
Column information such as following table
Note: joint sequence, which is that illumina microarray dataset institute is general, obtains joint sequence, as the differentiation between sample.
A kind of embodiment 2: STR classification system and its kit for complicated Relationship iden- tification
The chain str locus seat of 2.1 autosomes constitutes and characteristic:
(1) the STR classification system in the present embodiment includes following 58 str locus seats:
D1S3736、D1S1665、D1S2127、D1S2138、D1S1642、D1S518、D1S1604、D1S3729、
D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、D2S2944、D2S1327、D3S2402、
D3S1766、D4S2411、D4S3351、D4S243、D4S2426、D4S2373、D5S1490、D5S2856、D5S2495、
D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、D7S1799、D8S2324、D8S2330、
D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、D11S1983、D11S2363、D11S1368、
D12S393、D12S1063、D13S1807、D13S1492、D14S738、D14S301、D14S583、D15S816、D15S1514、
D18S872,D18S972,D18S548,D20S1145,D20S477；
It is with following measurable properties:
1) core space recurring unit is tetranucleotide；
2) heterozygosity of each STR is greater than 0.6；
3) every group of STR number at least two for including and its genetic distance are less than 3cM；
4) each STR has the name of DXSXX.
(2) it is used for while expanding the primer combination 2 of 58 str locus seats in the present embodiment are as follows:
Table 3: the forward primer group of primer combination 2
Table 4: the reverse primer group of primer combination 2
(3) working concentration of forward primer is 0.1 μM in the present embodiment；The working concentration of reverse primer is 0.1 μM.
A kind of 2.2 kits for complicated Relationship iden- tification:
Difference with embodiment 1 is only that PCR primer combination used is primer combination 2.
A kind of embodiment 3: STR classification system and its kit for complicated Relationship iden- tification
The chain str locus seat of 3.1 autosomes constitutes and characteristic:
(1) the STR classification system in the present embodiment includes following 61 str locus seats:
D1S532、D1S1611、D1S3733、D1S533、D1S1614、D2S2977、D2S1374、D2S1394、
D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、D3S2388、D4S1626、D4S1653、
D5S2858、D5S2796、D5S1463、D5S815、D5S2499、D5S2498、D6S1043、D6S1274、D6S1056、
D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、D8S2326、D8S1464、D8S2320、
D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、D10S2469、D10S1238、D11S4464、
D11S4958、D13S1818、D13S767、D14S615、D14S608、D14S597、D14S302、D14S749、D16S767、
D16S3393,D18S537,D18S875,D18S1367,D20S1152,D20S206,D20S607,D20S1146；
It is with following measurable properties:
1) core space recurring unit is tetranucleotide；
2) heterozygosity of each STR is greater than 0.6；
3) every group of STR number at least two for including and its genetic distance are less than 3cM；
4) each STR has the name of DXSXX.
(2) it is used for while expanding the primer combination 3 of 61 str locus seats in the present embodiment are as follows:
Table 5: the forward primer group of primer combination 3
Table 6: the reverse primer group of primer combination 3
(3) working concentration of forward primer is 0.1 μM in the present embodiment；The working concentration of reverse primer is 0.1 μM.
A kind of 3.2 kits for complicated Relationship iden- tification:
Difference with embodiment 1 is only that PCR primer combination used is primer combination 3.
A kind of embodiment 4: STR classification system and its kit for complicated Relationship iden- tification
The chain str locus seat of 4.1 autosomes constitutes and characteristic:
(1) the STR classification system in the present embodiment includes 118 str locus seats, specifically includes described in embodiment 1 60
A str locus seat and 58 str locus seats as described in example 2.
It is with following measurable properties:
1) core space recurring unit is tetranucleotide；
2) heterozygosity of each STR is greater than 0.6；
3) every group of STR number at least two for including and its genetic distance are less than 3cM；
4) each STR has the name of DXSXX.
(2) it is used for while the primer combination 4 for expanding 118 str locus seats in the present embodiment is embodiment 1 and embodiment
The combination that primer combination in 2 is constituted.
(3) working concentration of forward primer is 0.1 μM in the present embodiment；The working concentration of reverse primer is 0.1 μM.
A kind of 4.2 kits for complicated Relationship iden- tification:
Difference with embodiment 1 is only that PCR primer combination used is primer combination 4.
A kind of embodiment 5: STR classification system and its kit for complicated Relationship iden- tification
The chain str locus seat of 5.1 autosomes constitutes and characteristic:
(1) the STR classification system in the present embodiment includes 121 str locus seats, specifically includes described in embodiment 1 60
A str locus seat and 61 str locus seats described in embodiment 3.
It is with following measurable properties:
1) core space recurring unit is tetranucleotide；
2) heterozygosity of each STR is greater than 0.6；
3) every group of STR number at least two for including and its genetic distance are less than 3cM；
4) each STR has the name of DXSXX.
(2) it is used for while the primer combination 5 for expanding 121 str locus seats in the present embodiment is embodiment 1 and embodiment
The combination that primer combination in 3 is constituted.
(3) working concentration of forward primer is 0.1 μM in the present embodiment；The working concentration of reverse primer is 0.1 μM.
A kind of 5.2 kits for complicated Relationship iden- tification:
Difference with embodiment 1 is only that PCR primer combination used is primer combination 5.
A kind of embodiment 6: STR classification system and its kit for complicated Relationship iden- tification
The chain str locus seat of 6.1 autosomes constitutes and characteristic:
(1) the STR classification system in the present embodiment includes 119 str locus seats, specifically includes as described in example 2 58
A str locus seat and 61 str locus seats described in embodiment 3.
It is with following measurable properties:
1) core space recurring unit is tetranucleotide；
2) heterozygosity of each STR is greater than 0.6；
3) every group of STR number at least two for including and its genetic distance are less than 3cM；
4) each STR has the name of DXSXX.
(2) it is used for while the primer combination 6 for expanding 119 str locus seats in the present embodiment is embodiment 3 and embodiment
The combination that primer combination in 2 is constituted.
(3) working concentration of forward primer is 0.1 μM in the present embodiment；The working concentration of reverse primer is 0.1 μM.
A kind of 6.2 kits for complicated Relationship iden- tification:
Difference with embodiment 1 is only that PCR primer combination used is primer combination 6.
A kind of embodiment 7: STR classification system for complicated Relationship iden- tification
The chain str locus seat of 7.1 autosomes constitutes and characteristic:
(1) the STR classification system in the present embodiment includes 179 str locus seats, specifically includes described in embodiment 1 60
A str locus seat, 58 str locus seats as described in example 2 and 61 str locus seats described in embodiment 3.
It is with following measurable properties:
1) core space recurring unit is tetranucleotide；
2) heterozygosity of each STR is greater than 0.6；
3) every group of STR number at least two for including and its genetic distance are less than 3cM；
4) each STR has the name of DXSXX.
(2) it is used for while the primer combination 7 for expanding 179 str locus seats in the present embodiment is embodiment 1, embodiment 3
The combination constituted with the primer combination in embodiment 2.
(3) working concentration of forward primer is 0.1 μM in the present embodiment；The working concentration of reverse primer is 0.1 μM.
A kind of 7.2 kits for complicated Relationship iden- tification:
Difference with embodiment 1 is only that PCR primer combination used is primer combination 7.
Experimental example
In following experimental examples, used reagent is that legal commercially available approach can get, and it is as follows to obtain channel:
Tissue, blood DNA extracts kit: it is purchased from Beijing Tiangeng biochemical technology Co., Ltd；Article No. are as follows: DP304-03；
QIAamp DNA Investigator Kit (50): it is purchased from Kai Jie company, Germany；Article No. are as follows: 5650；
Enhancer buffer NB (1N): the PCR increased response agent purchased from the entitled NB of Ai Ji TAIKANG Company；Article No.
Are as follows: MT017035
IGT-EM707 polymerase mixture: Ai Ji TAIKANG Company, the archaeal dna polymerase of entitled EM707 are purchased from
Mixed liquor；Article No. are as follows: MT017035；
YF Buffer B: the magnetic bead wash buffer purchased from the entitled YF of Ai Jitaikang；Article No. are as follows: MT017035；
Mentioned reagent is Beijing Ai Ji TAIKANG Company (iGeneTechTM) customized synthesis, article No. are as follows: MT017035
Primer or Index sequence: commission Beijing Ai Ji TAIKANG Company (iGeneTechTM) customized synthesis；
1, sample: 108 Han nationality in Beijing independent individuals samples, including 48 blood samples and 60 FTA blood cards is detected.
2, test object: being respectively adopted classification system described in embodiment 1-7 and kit, for detection sample according to inspection
Flow gauge is detected.
3, testing process:
(1) tissue, blood DNA extracts kit and QIAamp DNA is respectively adopted in blood sample, FTA blood card
Investigator Kit (50) extracts complete genome DNA.Concentration mensuration, quantitative genomic DNA are carried out using nucleic acid quantification instrument
Concentration is 1,5,10,20ng/ μ L.
(2) multiplex PCR library construction:
Using above-mentioned 1,5,10, the genomic DNA of 20ng/ μ L carry out library construction, bank number be respectively F01, F02,
F03 and F04.
A. reaction mixture, every 25 μ L of pipe, according to the reaction condition of table 8 first round multi-PRC reaction: are prepared according to table 7
Multi-PRC reaction is carried out, multiple PCR products are obtained；Wherein primer combination respectively refers to reality in different detection tests
Apply the primer combination in a 1-7；
Table 7: first round multi-PRC reaction system
| Reagent | Volume (μ L) |
| Distilled water | 4 |
| Enhancer buffer NB(1N) | 7 |
| Primer combination | 8 |
| Genomic DNA | 1 |
| IGT-EM707polymerase mixture | 5 |
Table 8: the condition of first round multi-PRC reaction
First round magnetic beads for purifying:
AMPure XP magnetic bead after 23 μ L equilibrium at room temperature are added to 25 μ L multiple PCR products is beaten with pipettor suction and mixes number
It is secondary, after being incubated at room temperature 5min, PCR pipe is placed in 3min on DynaMag-96 Side magnetic frame, supernatant is thoroughly removed, by PCR pipe
It is removed from magnetic frame, 40 μ L YF buffer B (magnetic bead wash buffer) is added with pipettor suction and play mixing for several times, room temperature is incubated
5min is educated, supernatant is removed, 180 μ L, 80% ethanol solution is added, stands 30s, removes supernatant, it is molten that 180 μ L, 80% ethyl alcohol is added
Liquid thoroughly removes supernatant after standing 30s, is stored at room temperature 3min, residual ethanol is made thoroughly to volatilize, and 24 μ L Nuclease- are added
Free water (nuclease-free water) or 1 × TE buffer (pH 8.0), pipettor, which is gently inhaled to beat, is resuspended magnetic bead, avoids producing
Anger bubble, is stored at room temperature 2min, PCR pipe is replaced on magnetic frame and stands 3min, with pipettor Aspirate supernatant, be transferred to
In new PCR pipe, managing interior supernatant is multiple PCR products after purification.
B. second take turns multi-PRC reaction: the multiple PCR products after purification obtained using step A carry out the second wheel as template
Multi-PRC reaction is inserted into Index joint sequence, and the second wheel multi-PRC reaction system is as shown in table 9, reaction condition such as 10 institute of table
Show；
Table 9: the second takes turns multi-PRC reaction system
| Reagent | Volume (μ L) |
| Multiple PCR products after purification | 18 |
| IGT-I5 Index(10μM) | 1 |
| IGT-I7 Index(10μM) | 1 |
| IGT-EM707polymerase mixture | 5 |
Table 10: the second takes turns multi-PRC reaction condition
Second wheel magnetic beads for purifying:
Concrete operations are identical as first round magnetic beads for purifying step, and final step is transferred to the supernatant in new PCR pipe as system
The multiplex PCR library got ready.
C. quantitative and quality testing is carried out to obtained multiplex PCR library:
The multiplex PCR library for taking 1 μ L step B to obtain carries out library concentration measurement using nucleic acid quantification instrument, and record library is dense
Degree；
It is long to carry out library fragments using full-automatic nucleic acid-protein analysis system for the multiplex PCR library for taking 1 μ L step B to obtain
The target segment distributed area of degree and purity detecting, normal library is 300bp-450bp, and main peak is in 339bp or so.
(3) sequencing of two generations is carried out to the multiplex PCR library that step (2) obtains, the fastq file that sequencing obtains is used
FASTQC software carries out quality control, is then filtered with Trimmomatic software to data, and trimming is lower than the sequence of Q30,
Remove sequencing fragment be lower than 100bp sequence, then by the fastq file after Quality Control with STRaitRazor3.0 to STR's
Parting will finally obtain STR parting file corresponding to each individual.
4, testing result:
(1), parting accurately can be realized for 108 samples using classification system disclosed in embodiment 1-7 and kit
And Relationship iden- tification.
(2), the quality inspection peak figure difference in the library of preparation is combined using primer disclosed in embodiment 1-7 as shown in figs. 1-7: table
Bright 7 STR classification systems provided by the invention and its kit can expand above-mentioned 60 disposably, simultaneously, 58,61,118,
121,119 or 179 str locus seats, and balance is good, primer specificity is good, high sensitivity, can effectively detectable concentration down to
The gDNA of 1ng/ μ L.
(3), can effectively be divided using classification system disclosed in embodiment 1-7 and kit for 108 sample standard deviations
Type information is Rational Simplification application documents, now enumerates the parting information of wherein 3 samples:
Analyze software: 3.0 software of STRait Razor
Table is explained: following blank cell represents the site of the sample as homozygote, and two kinds of partings represent heterozygote；Each
The subsequent sample reads number of parting, which is represented, obtains corresponding point of the site obtained by the configuration file using STRait Razor 3.0
The coverage of type.
Genotyping result information of the STR bit o'clock in 3 samples in embodiment 1 is as follows:
Genotyping result information of the STR bit o'clock in 3 samples in embodiment 2 is as follows:
Genotyping result information of the STR bit o'clock in 3 samples in embodiment 3 is as follows:
The result that embodiment 4 is equal to embodiment 1 and embodiment 2 to the genotyping result of above-mentioned 3 samples merges；Embodiment
The genotyping result of 5 pairs of above-mentioned 3 samples is equal to embodiment 1 and the result of embodiment 3 merges；Embodiment 6 is to above-mentioned 3 samples
Genotyping result be equal to the result of embodiment 2 and embodiment 3 and merge；Embodiment 7 is equivalent to the genotyping result of above-mentioned 3 samples
Merge in the result of embodiment 1, embodiment 2 and embodiment 3；To avoid repeating, it is not repeated to place table herein.
(4), parting efficiency:
60,58,61,118,121,119,179 separately included in classification system disclosed in embodiment 1-7 and kit
The medical jurisprudence parameter of STR bit point is as follows:
Analysis tool: STRAF 1.0.5:(STR Analysis for Forensics)；
Analyze data source: the genotyping result information of above-mentioned 108 Han nationality in Beijing independent individuals sample；
Table is explained: the name of Locus expression str locus seat；N indicate allele number, totally 108 Han nationality in Beijing without
Individual is closed, therefore each STR genetic marker there are 216 allele；Nall indicates the sum of each locus parting, the model of parting
It encloses for 5-19；GD (Genetic diversity, genetic polymorphism), in the range of between 0.4911-0.9013；PIC
(Polymorphism Information Content, polymorphism information content), in the range of between 0.4368-0.8880；
PM (match probability, matching probability), range is between 0.0297-0.5111；PD(power of
Discrimination, personal identification probability), range is between 0.4889-0.9703.
(5), library concentration:
GDNA initial amount RFU (fluorescence signal intensity) value in 1ng and 5ng is slightly relatively low, but is normal main peak, when
In 10ng and 20ng, not only library is mainly normal main peak to gDNA initial amount but also RFU value is more preferable.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of compound parting system for Relationship iden- tification, which is characterized in that the compound parting system includes using
In the PCR primer combination 2 for expanding 58 chain euchromosome STR locus；
The chain euchromosome STR locus of described 58 be D1S3736, D1S1665, D1S2127, D1S2138, D1S1642,
D1S518、D1S1604、D1S3729、D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、
D2S2944、D2S1327、D3S2402、D3S1766、D4S2411、D4S3351、D4S243、D4S2426、D4S2373、
D5S1490、D5S2856、D5S2495、D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、
D7S1799、D8S2324、D8S2330、D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、
D11S1983、D11S2363、D11S1368、D12S393、D12S1063、D13S1807、D13S1492、D14S738、
D14S301,D14S583,D15S816,D15S1514,D18S872,D18S972,D18S548,D20S1145,D20S477；
The PCR primer combination 2 includes forward primer and reverse primer；
The forward primer is as follows:
The reverse primer is as follows:
2. compound parting system according to claim 1, which is characterized in that the compound parting system further includes using
In the PCR primer combination 3 for expanding 61 chain euchromosome STR locus；
The chain euchromosome STR locus of described 61 be D1S532, D1S1611, D1S3733, D1S533, D1S1614,
D2S2977、D2S1374、D2S1394、D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、
D3S2388、D4S1626、D4S1653、D5S2858、D5S2796、D5S1463、D5S815、D5S2499、D5S2498、
D6S1043、D6S1274、D6S1056、D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、
D8S2326、D8S1464、D8S2320、D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、
D10S2469、D10S1238、D11S4464、D11S4958、D13S1818、D13S767、D14S615、D14S608、D14S597、
D14S302、D14S749、D16S767、D16S3393、D18S537、D18S875、D18S1367、D20S1152、D20S206、
D20S607,D20S1146；
The PCR primer combination 3 includes forward primer and reverse primer；
The forward primer is as follows:
The reverse primer is as follows:
3. compound parting system according to claim 2, which is characterized in that the compound parting system further includes using
In the PCR primer combination 1 for expanding 60 chain euchromosome STR locus；
The chain euchromosome STR locus of described 60 be D1S2131, D1S3721, D1S2130, D1S1600, D1S1653,
D1S1660、D1S3732、D2S1364、D2S2734、D2S1396、D2S428、D2S435、D2S1387、D2S1792、
D2S1399、D2S2959、D3S2431、D3S4547、D4S1643、D4S2408、D4S3326、D4S2368、D5S2845、
D5S1473、D5S813、D5S1716、D5S1459、D5S1487、D5S1466、D5S2496、D5S2501、D6S1019、
D6S2417、D6S2412、D6S1284、D7S1820、D7S3050、D7S821、D8S594、D8S1468、D8S2322、D8S569、
D8S1475、D9S746、D9S319、D9S2149、D11S2010、D11S1392、D12S376、D12S1052、D13S317、
D13S790, D14S1432, D14S1428, D15S644, D15S1507, D16S752, D16S485, D20S481 and D20S1151；
The PCR primer combination 1 includes forward primer and reverse primer；
The forward primer is as follows:
The reverse primer is as follows:
4. any more than 30 str locus seats and its forward direction in the str locus seat as described in any one of claim 1-3
The STR classification system constituted with reverse primer.
5. a kind of kit for complicated Relationship iden- tification, which is characterized in that the kit includes claim 1 institute
That states is used to expand the PCR primer combination 2 of 58 chain euchromosome STR locus, forward direction in the PCR primer combination 2
The working concentration of primer and reverse primer is 0.1 μM.
6. kit according to claim 5, it is characterised in that: further include as claimed in claim 2 for expanding 61
The PCR primer combination 3 of chain euchromosome STR locus, forward primer and reverse primer in the PCR primer combination 3
Working concentration is 0.1 μM.
7. kit according to claim 6, it is characterised in that: further include as claimed in claim 3 for expanding 60 companies
The PCR primer combination 1 of euchromosome STR locus is locked, the work of forward primer and reverse primer in the PCR primer combination 1
Making concentration is 0.1 μM.
8. according to kit described in claim 5-7 any one, which is characterized in that the kit further includes Index
Joint sequence and archaeal dna polymerase.
9. kit according to claim 8, which is characterized in that the Index joint sequence includes IGT-I5
Index and IGT-I7 Index, working concentration are 10 μM.
10. kit according to claim 8, which is characterized in that the kit further includes that genomic DNA is made
For the reagent in the library of sequencing.
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