CN1327005C - Complex aplification detecting system of fluorescent marker short tandem repetitive sequence gene locus - Google Patents
Complex aplification detecting system of fluorescent marker short tandem repetitive sequence gene locus Download PDFInfo
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- CN1327005C CN1327005C CNB2004100966136A CN200410096613A CN1327005C CN 1327005 C CN1327005 C CN 1327005C CN B2004100966136 A CNB2004100966136 A CN B2004100966136A CN 200410096613 A CN200410096613 A CN 200410096613A CN 1327005 C CN1327005 C CN 1327005C
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Abstract
The present invention provides a fluorescence labeling verification system by compounding and expanding 14 short series repeating sequence locus to take personal identification and paternity tests, such as FAM: D2S1338, D6S1043, D21S11, D7S820, CSF1PO; JOE: D3S1358, D8S1179, D16S539, PentaE; TMAR: D5S818, vWA, D13S317, D18S51 and FGA. The gene locus is justified with height genetic polymorphism in Chinese population. The specific compounding method controls expanding products in a range of 400 basic groups (bp), and the compounding expanding of the 14 gene locus can be realized via only labeling three types of fluorescence. The present invention takes a deep research to gene characteristic of the 14 locus and allele thereof in five minorities in China, provides an allele standard control system and a complex amplification detecting system of fluorescent labeling short tandem repeating sequence gene locus and research an agent box which is suitable for Chinese people.
Description
Technical field
The present invention relates to have in the human body genome genetic marker of polymorphism.The present invention be more particularly directed to polymerase chain reaction a plurality of short tandem repeats that in a reaction, increase simultaneously.The invention still further relates to a kind of fluorescent mark STR composite amplification checking system that carries out individual's identification and paternity test by 14 str locus seats of composite amplification.Checking system of the present invention is fit to Chinese population.
Background technology
STR locus (STR) is the genetic marker of present widespread usage.The beginning of the nineties str locus seat polymorphism discovery, particularly the str locus seat has the little easy amplification of fragment, be suitable for check trace and degraded sample, and the amplification condition of each locus is similar and can composite amplification, thus have sensitivity, accurately, fast, advantage such as contain much information.Especially setting up aspect the DNA database, STR composite amplification technology has great superiority.Therefore U.S. FBI has selected 13 str locus seats to be used to set up database---CODIS (Combined DNA Index System): CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, HUMTH01, TPOX, vWA.These str locus seats are commonly called 13 core gene seats.Just because of advantage that STR possessed and application prospect, legal medical expert educational circles and the company that some are big all drop into the research of a large amount of funds to its developing property.
The middle and later periods nineties is that the research and development merchant of representative sets up the fluorescent composite amplification system with American AB I company.The most representative at present is ABI (Applied Biosystems, USA) Identifiler of company
TMAnd the PowerPlex16 of Promega (USA) company
The fluorescent reagent box.These two test kits include above-mentioned 13 core gene seats.
Secular DNA check practice shows that there is certain difference in the genetic polymorphism of STR locus between the race, and difference is also very big between each locus.In 13 core gene seats that U.S. FBI recommends, have portion gene seat genetic polymorphism not high, or the different crowd differences is bigger.Bring certain influence for thus the application and the efficient thereof of DNA inspection technology.
We have carried out deep research to the genetic polymorphism of the STR locus of Chinese population, and develop the composite amplification checking system of new STR locus on this basis, broken through the pattern of existing 13 core gene seats (FBI recommendation), improve the DNA efficiency of test, developed the compound checking system of fluorescence STR simultaneously.
Summary of the invention:
The present invention relates to have in the human body genome genetic marker of polymorphism.The present invention be more particularly directed to polymerase chain reaction a plurality of short tandem repeats that in a reaction, increase simultaneously.The invention still further relates to a kind of fluorescent mark STR composite amplification checking system that carries out individual's identification and paternity test by 14 str locus seats of composite amplification.
14 STR locus that the present invention is fit to Chinese population constitute: D2S1338, D6S1043, D21S11, D7S820, CSF1PO, D3S1358, D8S1179, D16S539, PentaE, D5S818, vWA, D13S317, D18S51 and FGA.The present invention has carried out deep research to above-mentioned 14 STR locus and allelotrope hereditary feature thereof, shows that these 14 STR locus have the genetic polymorphism of height in Chinese population and good gene frequency distributes.
Above-mentioned 14 short series connection being repeated the serial genes seats in actual detection adds and is used to differentiate that other Amelogenin locus of human nature forms 15 composite amplification checking systems that locus detects simultaneously.
The invention provides a kind of fluorescent mark STR composite amplification checking system that carries out individual's identification and paternity test by 15 locus of composite amplification, its fluorescence array mode is that FAM is fluorescein-labelled: D6S1043+D21S11+D7S820+CSF1PO+D2S1338; JOE is fluorescein-labelled: D3S1358+D13S317+D8S1179+D16S539+PentaE; TAMRA is fluorescein-labelled: Amelogenin+D5S818+vWA+D18S51+FGA.Because this specific array mode is controlled at amplified production in 400 bases (bp) scope.This locus constitutes, and has broken through the pattern of existing 13 core gene seats (FBI recommendations), the technical requirements and the population genetics feature of Chinese population of coincidence method medical science DNA check more, and taken into account international exchange and shared standard requirement.
The invention provides the locus unitized design scheme of fluorescent mark STR composite amplification system.The present invention has selected for use Fluoresceincarboxylic acid as the blue-fluorescence labelled reagent; Select tetramethyl--6 carboxyl rhodamine (TAMRA) and chlordene fluorescein (JOE) respectively as yellow and green fluorescence labelled reagent simultaneously, interior mark is selected CXR for use, has made up 4 look fluorescence assembled schemes.The locus array mode of this original creation and design of primers can only need three kinds of fluorescence of mark just can realize the composite amplification of these 15 locus in same reaction, full gene seat amplified production is controlled at realizes highly sensitive in 400 bases (bp) scope simultaneously.
The invention provides a kind of fluorescence labeling composite amplification system of a plurality of str locus seats of analyzing gene group DNA simultaneously that is used for, comprise the Oligonucleolide primers of 15 locus that are used for the composite amplification analysis, the primer of whole 15 locus is blended in one in vitro.Locus in the composite amplification system is positioned at a pair of primer amplification of these locus both sides, wherein has 5 ' end of a primer to carry out fluorescein-labelled in every pair of primer.
The invention provides a kind of allelotrope standard control objects system corresponding with the DNATyper15 system.
The present invention at first develops 15 locus, four look fluorescent mark STR composite amplification checking systems (DNATyper15) of the composite amplification in single reaction simultaneously at home, reaches at present the highest level of fluorescent mark STR composite amplification reagent kit in the world.
Its good effect mainly shows following several aspect:
1. the sensitivity of system
The DNATyper15 fluorescent mark STR composite amplification checking system that the present invention developed is under the condition of 0.125ng in the dna profiling amount, can detect whole 15 locus.
2. irrelevant individual dependent probability
With DNATyper15 fluorescent mark STR composite amplification checking system to Chinese Chinese Han population more than totally 300 sample result of carrying out the genetics investigation show: this reagent in Chinese han population totally at random matching probability be 3.49 * 10
-18, its accumulation parentage exclusion probability is 0.9999996.
3. the effect of practical application
The DNATyper15 fluorescent mark STR composite amplification system that the present invention developed is on probation in the case inspection routine of the public security system of 10 provinces and cities at home, result on trial shows this system's polymorphism height, balance is better, highly sensitive, the somatotype result is accurate, can satisfy the needs of actual case check fully.
Detailed Description Of The Invention
One, locus determines
Existing str locus seat is analysed in depth research and preferred new fine resolution locus.24 str locus seats such as D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D13S317, D16S539, TPOX, TH01, D5S818, vWA, D18S51, FGA, D2S1338, D19S433, PentaE, PentaD, D19S253, D12S391, D1S1612, D9S302, D18S535, D15S816, D6S1043 are carried out investigation in the genetic polymorphism of Chinese population.Research has been established the str locus seat and has been constituted, i.e. D2S1338, D6S1043, D21S11, D7S820, CSF1PO, D3S1358, D8S1179, D16S539, PentaE, D5S818, vWA, D13S317, D18S51 and FGA.This locus constitutes, and has broken through the pattern of existing 13 core gene seats (FBI recommendation), more the technical requirements of coincidence method medical science DNA check and the population genetics feature of Chinese population.
Two, the locus assembled scheme of fluorescent mark STR composite amplification system design
This research has been carried out discriminating, has been selected fluorescence dye, has selected for use Fluoresceincarboxylic acid as the blue-fluorescence labelled reagent; Select tetramethyl--6 carboxyl rhodamine (TAMRA) and chlordene fluorescein (JOE) respectively as yellow and green fluorescence labelled reagent simultaneously, interior mark is selected CXR for use, has made up 4 look fluorescence assembled schemes.
On the basis of determining 4 look fluorescence assembled schemes, by experiment repeatedly in a large number, design 15 locus array modes at home first voluntarily: blue (FAM) mark D6S1043, D21S11, D7S820, CSF1PO and D2S1338; Green (JOE) mark D3S1358, D13S317, D8S1179, D16S539 and PentaE; Yellow (TAMRA) mark Amelogenin, D5S818, VWA, D18S51 and FGA, and design the primer sequence of 15 str locus seats voluntarily according to each locus position.
The locus array mode of this original creation and design of primers can only need three kinds of fluorescence of mark just can realize the composite amplification of these 15 locus in same reaction, full gene seat amplified production is controlled at realizes highly sensitive in 400 bases (bp) scope simultaneously.
The present invention at first develops 15 locus, four look fluorescent mark STR composite amplification checking systems (DNATyper15) of the composite amplification in single reaction simultaneously at home, reaches at present the highest level of fluorescent mark STR composite amplification reagent kit in the world.
Three, the optimization of fluorescent mark STR composite amplification system and foundation
1, the allotment of quality of balance between the locus
Along with the increase of complex locus number, because the influence of competition, the relative equilibrium control difficulty of each locus strengthens, and by experiment repeatedly repeatedly, constantly regulates primer concentration and proportioning, reaches balance at last.
2, the foundation of composite amplification condition
Single amplification condition to 15 locus is optimized earlier, on the basis of successfully having set up individual gene seat amplification condition, study 15 locus composite amplification PCR reaction conditionss, determined each parameter in the composite amplification by a large amount of experiments repeatedly, as, the variation of loop parameter, annealing temperature, damping fluid ionic strength, enzyme amount, composite amplification reaction volume and template DNA amount etc., make amplified production reach balance, special requirement, set up composite amplification system, amplify 15 locus simultaneously.
Description of drawings
Fig. 1 is with the STR somatotype result of aforesaid method check to irrelevant individual DNA sample;
Fig. 2 is the allelic ladder of full gene seat in the DNATyper15 system;
Fig. 3 is for detecting the figure as a result of a routine father-son-female triplet parent with the method for embodiment 1.
Embodiment
Embodiment 1
The composite amplification of DNATyper15 fluorescent mark STR composite amplification system and the fluoroscopic examination mankind's STR
Genotype
1, pcr amplification system
DNATyper15 5 * reaction solution 4.0 μ l
DNATyper15 primer mixture 2.0 μ l
Gold Taq DNA polysaccharase 0.4 μ l
Template DNA sample 1 μ l
Water 12.6 μ l
Total reaction volume 20 μ l
2, amplification thermal cycling
(1) the pcr amplification pipe is placed on the thermal cycler.
(2) program of selecting to recommend is below carried out thermal cycling.
(3) sample after the amplification should keep in Dark Place for a long time at-20 ℃,
The amplification program of 9600 and 9700 thermal cyclers
| Initial sex change | Sex change | Annealing | Extend | Extend eventually | Insulation |
| 30 circulations | |||||
| 95 ℃ 11 minutes | 94 ℃ 1 minute | 59 ℃ 1 minute | 72 ℃ 1 minute | 60 ℃ 45 minutes | Keep for 25 ℃ |
3, amplified production is with 310 or 3100 genetic analyzer fluoroscopic examinations
(1) uses ABI PRISM
310 genetic analyzers detect amplified production
1. the preparation of sample
The last sample mixture of forming by interior mark 600 (ILS 600) and deionized formamide: [(0.35 μ lILS600) * (sample introduction number)]+[(12.0 μ l deionized formamide) * (sample introduction number)], mix.The amplified production of 12 μ l mixtures and 1.0 μ l is mixed.Sample in the preparation.
2. instrument setting
Allelic Ladder in sample and the DNATyper15 system 95 ℃ of sex change 3 minutes, was placed on cooled on ice 3 minutes immediately, electrophoresis as early as possible after the sex change.
(2) use ABI PRISM
3100 DNA genetic analyzers detect the amplified production sample and prepare
Press the last sample mixture that following method preparation is made up of interior mark 600 (ILS 600) and deionized formamide: [(0.35 μ l ILS 600) * (sample introduction number)]+[(20.0 μ l deionized formamide) * (sample introduction number)], mix.The amplified production of 15 μ l mixtures and 1.0 μ l is mixed.Sample in the preparation.
With 95 ℃ of sex change of sample 3 minutes, be placed on cooled on ice immediately 3 minutes.Afterwards as early as possible at ABI PRISM
Electrophoresis on 3100 genetic analyzers.
Embodiment 2
Use DNATyper15 fluorescent mark STR composite amplification system and carry out paternity test.
1, principle:
According to mendel's law, parental generation genotype determiner is for genotype.Under the prerequisite that does not have transgenation, somatotype mistake, the ultimate principle of authentication method can reduce 2 points: 1. child's pair of alleles must be one from father, one from mother; 2. child can not have the allelotrope that parents all do not have.That is to say that at sure certain allelotrope of child be own father's gene, and when supposing that the father is not with this equipotential gene, can get rid of him and be child's own father; Come from its father at sure certain allelotrope of child, and the hypothesis father can not get rid of him and be child's own father when also having this equipotential gene.If compare the analysis difference of sample, can get rid of their sibship, if identical, then can not get rid of.Suppose that in theory not meeting genetic development between father and the child on a genetic marker can get rid of its set membership, but because the existence of sudden change, especially the high mutation rate of microsatellite marker (STR), the eliminating of a locus, may cause by sudden change, therefore not being inconsistent of a genetic marker can not be got rid of its parent, and requiring to get rid of simultaneously at 3 different genes seats could negate its parent.
In most of the cases, mother-child relationship (MCR) is known, require to identify whether one's own relation of father and child.
Under the situation that the genetic marker of all checks is not all got rid of, generally need to calculate the patriarchy probability.
2, the calculating of paternity index (PI):
Paternity index (PI) is the parent index again, is meant that controlled father possesses essential allelotrope and becomes the chance of biological parenthood (X) and possesses the ratio that essential allelotrope becomes biology father chance (Y) with man at random.
PI＝X/Y＝f×c/f×g
F represents that breeder mother gives child essential allelotrope chance
C represents that controlled father gives child essential allelotrope chance
G represents that at random the man gives child essential allelotrope chance, equals must each not chain total PI in site of gene frequency to equal the product of each site PI.
3, the relative chance of set membership (RCP)
The relative chance of patriarchy is named parent relative chance again, and parent probability or W value are represented the chance of controlled father as the biology father.
Suppose that controlled father is the child own father or be not that child own father's prior probability is identical that at this moment RCP and PI have following relation:
RCP＝(PI/PI+1)×100％
Total (PI/PI+1) * 100% of total RCP=
4, paternity test identification
The tradition genetic marker, paternity test identification standard is RCP 〉=99.73% in the world wide.Enter the DNA epoch, do not seek unity of standard both at home and abroad, Sichuan Province higher people's court legislation paternity test identification standard is RCP 〉=99.95%.
5, paternity test is got rid of
If the father-son, the mother-son that detect have 〉=and 3 above sites do not meet mendel's law, then get rid of parent.
Claims (9)
1. the fluorescence labeling composite amplification system of a plurality of str locus seats of a while analyzing gene group DNA is characterized in that: composite amplification analysis 15 locus: D2S1338, D6S1043, D21S11, D7S820, CSF1PO, D3S1358, D8S1179, D16S539, PentaE, D5S818, vWA, D13S317, D18S51, FGA and Amelogenin.
2. composite amplification system according to claim 1,15 locus simultaneously wherein increase in a composite amplification reaction system.
3. composite amplification system according to claim 1, wherein 15 locus are fluorescein-labelled by three kinds respectively in this system, and its locus divides three groups to make up, and its array mode is respectively: D6S1043+D21S11+D7S820+CSF1PO+D2S1338; D3S1358+D13S317+D8S1179+D16S539+PentaE; Amelogenin+D5S818+vWA+D18S51+FGA.
4. composite amplification system according to claim 3, wherein locus is positioned at a pair of primer amplification of these locus both sides, wherein has 5 ' end of a primer to carry out fluorescein-labelled in every pair of primer.
5. composite amplification system according to claim 1 wherein comprises the mixture of the pairing allelotrope standard substance of each locus in this system, to determine the allelotrope of each locus in the DNA sample.
6. according to the composite amplification system of claim 1, wherein the locus series of Xuan Zeing comprises is used to differentiate that the locus of DNA sample sex is the Amelogenin locus.
7. the method for a composite amplification is characterized in that: adopt the composite amplification system of claim 1, wherein a plurality of str locus seats of composite amplification are to carry out with polymerase chain reaction, and its detection method adopts multiple tracks or single track capillary gel electrophoresis.
8. the method for an analyzing gene group DNA is characterized in that application rights requires 1 to 3 described composite amplification system to come analyzing DNA.
9. method according to claim 8, wherein the DNA sample comprises seminal stain, salivary stain, tissue, blood stain or blood.
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| CNB2004100966136A CN1327005C (en) | 2004-12-03 | 2004-12-03 | Complex aplification detecting system of fluorescent marker short tandem repetitive sequence gene locus |
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| CNB2004100966136A CN1327005C (en) | 2004-12-03 | 2004-12-03 | Complex aplification detecting system of fluorescent marker short tandem repetitive sequence gene locus |
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| CN1327005C true CN1327005C (en) | 2007-07-18 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101818192B (en) * | 2009-08-27 | 2013-03-20 | 基点认知技术(北京)有限公司 | Compound amplification kit of 20 short tandem repeats |
| CN102559878B (en) * | 2011-12-21 | 2013-09-25 | 北京阅微基因技术有限公司 | Composite amplification system and detection kit of mouse short tandem repeat |
| CN105420400A (en) * | 2016-01-11 | 2016-03-23 | 中国科学院北京基因组研究所 | ZNA primer for trace degraded bio-sample miniSTR analysis |
| CN106011229B (en) * | 2016-04-26 | 2019-08-27 | 深圳华大法医科技有限公司 | The composite amplification systems of 18 STR bit points for people, kit and application thereof |
| CN107012225B (en) * | 2017-04-20 | 2020-10-09 | 司法鉴定科学研究院 | STR locus detection kit and detection method based on high-throughput sequencing |
| CN109337985A (en) * | 2018-07-06 | 2019-02-15 | 广州复能基因有限公司 | A kind of composite amplification system and kit of microsatellite instability detection in Gene Mutation |
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| CN1332805A (en) * | 1998-11-25 | 2002-01-23 | 普罗梅加公司 | Multiplex amplification of short tandem repeat loci |
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2004
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1219972A (en) * | 1996-04-15 | 1999-06-16 | 普罗梅加公司 | Multiplex amplification of short tandem repeat loci |
| CN1332805A (en) * | 1998-11-25 | 2002-01-23 | 普罗梅加公司 | Multiplex amplification of short tandem repeat loci |
| US6531282B1 (en) * | 2000-05-30 | 2003-03-11 | Oligotrail, Llc | Multiplex amplification and analysis of selected STR loci |
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Non-Patent Citations (3)
| Title |
|---|
| Evaluation of STR informativity for chimerism testing -comparative analysis of 27 STR systems in 203 matchedrelated donor recipient pairs C Thiede et al,Leukemia,Vol.Volume 18 No.Number 2 2004 * |
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