CN104046618B - New short nucleotides tandem repetitive sequence site and its application - Google Patents
New short nucleotides tandem repetitive sequence site and its application Download PDFInfo
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Abstract
The invention discloses a kind of new short nucleotides tandem repetitive sequence site and its application, tandem repeat loci G5S0003, sequence such as SEQ ID NO:Shown in 1, it is used for reagent or kit that genetic affinity is analyzed for preparing (a)；(b) it is used for the reagent or kit of individual identification；(c) it is used for the reagent or kit of paternity test or consanguinity analysis；(d) it is used to detect reagent or kit with the presence or absence of female blood stains dye in extracting amniotic fluid；And/or (e) be used to detecting leucocyte after bone-marrow transplantation in acceptor whether the kit replaced by donorcells.Tandem repeat loci G5S0003 discriminations are high, can effectively analyze genetic affinity.
Description
Technical field
The present invention relates to a kind of short nucleotides tandem repetitive sequence site and its application.
Background technology
STR (Short Tandem Repeat.STR), also known as microsatellite DNA (microsatellite
DNA), it is a class recurring unit 2-6bp, number of repetition 10-60, strand of dna connection repetitive sequence of the fragment length in below 400bp；
Its producing cause is slip during DNA replication dna, or sliding chain and complementary strand bases mispairing in duplication and reparation, causes one
Or missing or the insertion of several recurring units.STR is with widely distributed in genome, allele is more, heterozygosity is high, parting
As a result stablize, STR heredity meets Meng get Er law of inheritances (Koreth, J., et al.1996.Microsatellites and
PCR ge-nomic analysis.J.Pathol.178:239-248.) the features such as, and multiple str locus seat joint-detection
When, individual identification power and parentage exclusion probability are high.Thus STR is marked as second generation heredity with features such as its genetic polymorphisms
Note is widely used in the fields such as medical science individual identification, the drafting of human inheritance's collection of illustrative plates, paternity test, legal medical material evidence examination, effectively
Promote forensic identification to exclude to individual identification from the individual of conventional material evidence sample to change comprehensively.
At present, the technology of individual identification is mainly detected using STR-PCR, to multiple str locus seat united typing, to determine
Parental right relation or individual identification etc., in experimental method all relative maturities, but in the selection of str locus seat, do not seek unity of standard.
Site used is not quite similar in the detection kit that domestic and international manufacturer provides.Early stage America and Europe Jia 1 from 10 str locus seats
Individual sex determining gene (E.A.Cotton, R.F.Allsop, J.F.Guest, R.R.E.Frazier, P.Koumi,
I.P.Callow,A.Seager,R.L.Sparkes,Validation of the SGM PlusTMsystem
for use in forensic casework,Forensic Sci.Int.112(2000)151–161.).Later with technology
Development, database increase, and comparison information difficulty is increased, and the selection of str locus seat is also being updated.It is now more powerful
Database CODIS (Combined DNA Index System) be by U.S. FBI be based on 13 str locus seat (D3S1358,
VWA, D16S539, D5S818, D7S820, CSF1PO, D13S317, TPOX, D8S1179, D21S11, D18S51, TH01,
FGA.) build, thus now most of STR detection kits manufacturers also on the basis of this 13 core genes seat increase or
Delete some sites with improve detection efficiency and accuracy rate (D.J.French, R.L.Howard, N.Gale, T.Brown,
D.G.McDowell,P.G.Debenhan,Interrogation of short tandem repeats using
fluorescent probes and melting curve analysis:A step towards rapid DNA
identity screening.Forensic Science.(2008)333-339).Such as Promega companies16System kits include 13 core gene seats, and add PentaD, PentaE and AMELO tri-
Individual locus；American AB I companiesPCR Amplification Kit are then 13 cores
Heart locus adds D2S1338, D19S433 and AMELO；Other two kit of its companySGM
PlusTMPCR Amplification Kit andPCR Amplification Kit have also been made
The increase and decrease of corresponding gene seat point.The unit of the similar detection kit of domestic production also has a lot, and for example the Ministry of Public Security two is promoted
DNA TyperTM15, middle dolantin joins the AGCU EX22STR fluorescence detection reagent kits of Bioisystech Co., Ltd, institute's detection base
Because seat increases to 22.
The advantage of the str locus seat of above kit selection is, based on 13 core gene gained public acceptance seats,
The information content that some other sites are obtained with abundant detection is added, the otherness between individual can be more comprehensively reflected, and then
Complete the work such as individual identification, patriarchy judgement.But in these locus used all it is not optimal selection, such as FGA bases
Have that sequence length is long because of seat, more than repeat unit but span is discontinuous, so that the compatibility with other sites is have impact on, it is unfavorable
Used in the multiple system of multiple locus joint-detections；D19S433 locus is then located at highly repetitive sequence in genome
Area, amplimer design is present compared with burden, and the stability in the site is not high, and what is provided during applied to individual identification can
It is not high with information quality and discrimination；For another example D21S11 locus is located at highly repetitive sequence region in genome except existing
The problem of outside, it was found that the CNV (situations of gene copy number variation are there may be in the locus region；Running into
During the improper pattern detection of gene copy number, the degree of accuracy of site STR partings can be reduced, the base is used in individual's identification
Because the effect of seat can also have a greatly reduced quality.With the foundation and the development of sequencing technologies of human genome map, with relatively low cost,
Sequence is resurveyed to personal full-length genome to be possibly realized, and then can filter out discrimination under larger information content within the faster time
Higher str locus seat, has promoted the selection and renewal applied to the str locus seat in the research such as individual identification.
Therefore, to overcome poor compatibility, there is CNV or positioned at defects such as highly repetitive sequence regions, this area still needs one
Plant the new STR with high discrimination.
The content of the invention
It is an object of the invention to provide a kind of new tandem repeat loci, it is located in the absence of CNV, and not
Highly repetitive sequence region, with high discrimination.
There is provided a kind of tandem repeat loci G5S0003 of separation, sequence such as SEQ for the first aspect of the present invention
ID NO.:Shown in 1.
The second aspect of the present invention is there is provided a kind of nucleotide sequence of separation, and the structure of the nucleotide sequence is as follows：
X1-X2-X3
In formula, X1 is SEQ ID NO.:1-800 in 2；
X2 is that the heredity containing >=2 repeat unit atag and/or atga and/or taga and/or tata and/or gata is more
State area；
X3 is SEQ ID NO.:925-1664 in 2.
The third aspect of the present invention there is provided the purposes of the tandem repeat loci G5S0003 described in first aspect,
For preparing：
(a) it is used for reagent or kit that genetic affinity is analyzed；
(b) it is used for the reagent or kit of individual identification；
(c) it is used for the reagent or kit of paternity test or consanguinity analysis；
(d) it is used to detect reagent or kit with the presence or absence of female blood stains dye in extracting amniotic fluid；And/or
(e) be used to detecting leucocyte after bone-marrow transplantation in acceptor whether the kit replaced by donorcells.
In another preference, described reagent includes (i) primer or primer pair；(ii) probe；(iii) nucleic acid chip.
In another preference, the sequence such as SEQ ID NO. of described primer pair:3 and SEQ ID NO.:Shown in 4.
There is provided the primer of a species-specific amplification tandem repeat loci G5S0003 for the fourth aspect of the present invention
It is right, the sequence such as SEQ ID NO. of described primer pair:3 and SEQ ID NO.:Shown in 4.
In another preference, the tandem repeat loci G5S0003 is as described in relation to the first aspect.
The fifth aspect of the present invention there is provided a kind of kit, including：
(a) it is used for specific amplification tandem repeat loci G5S0003 primer pair；
(b) operation instructions.
In another preference, the tandem repeat loci G5S0003 is as described in relation to the first aspect.
In another preference, the kit also includes one or more genes that (c) specific amplification is selected from the group
The primer pair of seat：
D3S1358、vWA、D16S539、D5S818、D7S820、CSF1PO、D13S317、TPOX、D8S1179、D21S11、
D18S51、TH01、FGA、D2S1338、D19S433、AMELO。
The sixth aspect of the present invention there is provided a kind of amplification system, including：
(a) it is used for specific amplification tandem repeat loci G5S0003 primer pair；With
(b) primer pair of optional, one or more locus that specific amplification is selected from the group：
D3S1358、vWA、D16S539、D5S818、D7S820、CSF1PO、D13S317、TPOX、D8S1179、D21S11、
D18S51、TH01、FGA、D2S1338、D19S433、AMELO；
And the primer pair (a) and optional primer pair (b) are located at same amplification system.
In another preference, the tandem repeat loci G5S0003 is as described in relation to the first aspect.
In another preference, described amplification system is composite amplification system, wherein, described primer pair (b) includes
1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, or 16 pairs of specific primers pair；And/or
The composite amplification system is the composite amplification system of fluorescence labeling, wherein, the fluorescence labeling refers to select down
One or more fluorescent labeling reagents of group mark the primer of the locus：FAM、HEX、VIC、NED、PET、TAMRA、ROX、
Fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, TET, TAMRA, JOE, BODIPY TMR, Oregon
Green, rhodamine is green, rhodamine is red, texas Red or Ya Jima are yellow.
There is provided a kind of amplification method, including step for the seventh aspect of the present invention：
In the amplification system described in the 6th aspect, using sample to be detected as template, carried out using PCR
The amplification of locus, so as to obtain locus amplifications product.
In another preference, methods described also includes：The step of being detected to the locus amplifications product.
In another preference, described detection is selected from the group：Capillary Electrophoresis, sequencing, hybridization.
In another preference, the testing sample is selected from the group：Blood, saliva, seminal fluid, sweat, amniotic fluid, bone or
Hair.
There is provided a kind of genetic affinity analysis method, including step for the eighth aspect of the present invention：
In the amplification system described in the 6th aspect, using sample to be detected as template, carried out using PCR
The amplification of locus, so as to obtain locus amplifications product；With
Wherein, the sample to be detected includes i in the sample of nucleic acid S1, S2 ... ..., Si respectively from Different Individual, formula
For >=2 positive integer；
(2) to from different sample of nucleic acid S1, S2 ... ..., Si locus amplifications product is detected, so as to obtain pair
Should be in the testing result of different sample of nucleic acid；
(3) testing result that step (2) is obtained is compared, so that it is determined that the genetic affinity between Different Individual.
In another preference, the genetic affinity analysis includes individual identification analysis, paternity test analysis, genetic connection
Analysis.
The invention provides a kind of new str locus seat, in the absence of CNV, and be not located at highly repetitive sequence region, it is miscellaneous
Right height, high resolution, compatibility are high, can be used with existing most of sites in same system, can effectively distinguish with
The genetic affinity of machine crowd.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 is the detection peak figure of the multiple STR bit points of male parent；
Fig. 2 is the detection peak figure of maternal multiple STR bit points；
Fig. 3 is the detection peak figure of the multiple STR bit points of filial generation；
Fig. 4 is the detection peak figure of the multiple STR bit points of affinity-less relation sample.
Embodiment
The present inventor, by largely screening, filters out a kind of new short series connection first by in-depth study extensively
Repetitive sequence locus G5S0003, containing >=2 repeat unit atag and/or atga and/or taga and/or tata and/or
Gata genetic polymorphism area.The tandem repeat loci discrimination reaches more than 0.8512, with a small amount of conventional STR bases
Because of seat combination, genetic affinity can be effectively distinguished, discrimination is up to more than 0.99.On this basis, the present invention is completed.
Tandem repeat loci
The present invention isolates a kind of new tandem repeat loci, STR bit point information such as table by largely screening
Shown in 1.
Table 1STR essential information
A kind of tandem repeat loci for separation that the present invention is provided, is named as G5S0003, sequence such as SEQ ID
NO.:Shown in 1.
The repeat unit of repeat number 31, i.e., 31.
G5S0003 sites exist the diversity of repeat unit, i.e. repeat unit may by atag, atga, taga, tata and
One or more units in gata are combined.
Number of alleles obtained by number of alleles, as experimental result statistics.
The nucleotide sequence for the separation that the present invention is provided, structure is as follows：
X1-X2-X3
In formula, X1 is SEQ ID NO.:1-800 in 2；
X2 is that the heredity containing >=2 repeat unit atag and/or atga and/or taga and/or tata and/or gata is more
State area；
X3 is SEQ ID NO.:925-1664 in 2.
In another preference, described X2 length is 50-150bp, preferably 60-130bp or 70-120bp, more preferably
Ground 80-110bp.
In another preference, described X2 be containing >=10 repeat unit atag and/or atga and/or taga and/
Or tata and/or gata genetic polymorphism area.
In another preference, described X2 be containing >=20 repeat unit atag and/or atga and/or taga and/
Or tata and/or gata genetic polymorphism area.
In another preference, X2 is SEQ ID NO.:Polynucleotides or its fragment shown in 1 (are included in 5' ends, 3' ends
And/or intermediate region is because lacking the fragment that one or more bases are formed).
The tandem repeat loci G5S0003 of the present invention, can be used in：
(a) reagent or kit analyzed for genetic affinity are prepared；
(b) reagent or kit for individual identification are prepared；
(c) reagent or the kit for paternity test or consanguinity analysis are prepared；
(d) prepare for detecting reagent or kit in extracting amniotic fluid with the presence or absence of female blood stains dye；And/or
(e) prepare for detect leucocyte after bone-marrow transplantation in acceptor whether the kit replaced by donorcells.
Described reagent also includes (i) primer or primer pair；(ii) probe；(iii) nucleic acid chip.It is preferred that described
The sequence of primer pair such as SEQ ID NO.:3 and SEQ ID NO.:Shown in 4.
The present invention also provides the primer pair of a species-specific amplification tandem repeat loci G5S0003, and sequence is such as
SEQ ID NO.:3 and SEQ ID NO.:Shown in 4.
One or two primer in described primer pair carries detectable.
Described detectable includes：Fluorescent marker or quantum dot-labeled thing.
The fluorescence labeling includes but is not limited to：FAM, HEX, VIC, NED, PET, TAMRA, ROX, fluorescein, FITC,
IRD-700/800, CY3, CY5, CY3.5, CY5.5, TET, TAMRA, JOE, BODIPY TMR, Oregon is green, rhodamine is green, sieve
Red bright red, texas Red or Ya Jima are yellow.Above-mentioned fluorescent marker is known in the art label, is commercially available commodity,
It can be obtained by buying.
Kit
The kit that the present invention is provided, including：
(a) it is used for specific amplification tandem repeat loci G5S0003 primer pair；
(b) operation instructions.
It is preferred that the sequence of described primer pair such as SEQ ID NO.:3 and SEQ ID NO.:Shown in 4.
The kit also includes the primer pair for one or more locus that (c) specific amplification is selected from the group：
D3S1358、vWA、D16S539、D5S818、D7S820、CSF1PO、D13S317、TPOX、D8S1179、D21S11、
D18S51、TH01、FGA、D2S1338、D19S433、AMELO。
In the kit, described each specific primer is pointed in same or different container.
In another preference, described kit also includes one or more following reagents：
(d) it is used for the reagent that PCR is expanded；
(e) it is used for the reagent of electrophoresis；
(f) it is used for the reagent for extracting DNA；
(g) standard items.
Amplification system and amplification method
The amplification system that the present invention is provided, including：
(a) it is used for specific amplification tandem repeat loci G5S0003 primer pair；With
(b) primer pair of optional, one or more locus that specific amplification is selected from the group：
D3S1358、vWA、D16S539、D5S818、D7S820、CSF1PO、D13S317、TPOX、D8S1179、D21S11、
D18S51、TH01、FGA、D2S1338、D19S433、AMELO；
And the primer pair (a) and optional primer pair (b) are located at same amplification system.
It is preferred that the sequence of described primer pair such as SEQ ID NO.:3 and SEQ ID NO.:Shown in 4.
In another preference, described amplification system is polymerase chain reaction PCR amplification system.
In another preference, described amplification system is liquid phase.
In another preference, described amplification system, which contains polymerase, dNTP etc., is used for the reagent that PCR polymerize.
Preferably, described amplification system is composite amplification system, wherein, described primer pair (b) includes 1,2,3,4,
5,6,7,8,9,10,11,12,13,14,15, or 16 pairs of specific primers pair.
It is preferred that the composite amplification system is the composite amplification system of fluorescence labeling, wherein, the fluorescence labeling refers to
The primer of the locus is marked from one or more fluorescent labeling reagents of the following group：FAM、HEX、VIC、NED、PET、
TAMRA, ROX, fluorescein, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, TET, TAMRA, JOE, BODIPY
TMR, Oregon is green, rhodamine is green, rhodamine is red, texas Red or Ya Jima yellow.
In another preference, 5 ' ends of a primer in each pair of primer carry fluorescence labeling.
The amplification method of the present invention, including step：
In above-mentioned amplification system, using sample to be detected as template, the expansion of locus is carried out using PCR
Increase, so as to obtain locus amplifications product.
Methods described also includes：The step of being detected to the locus amplifications product.It is preferred that described detection is adopted
With the method being selected from the group：Capillary Electrophoresis, sequencing, hybridization.
The testing sample is selected from the group：Blood, saliva, seminal fluid, sweat, amniotic fluid, bone or hair.
In another preference, the sample comes from people.
Genetic affinity analysis method
The present invention provides a kind of genetic affinity analysis method, including step：
(1) in above-mentioned amplification system, using sample to be detected as template, locus is carried out using PCR
Amplification, so as to obtain locus amplifications product；With
Wherein, the sample to be detected includes i in the sample of nucleic acid S1, S2 ... ..., Si respectively from Different Individual, formula
For >=2 positive integer；
(2) to from different sample of nucleic acid S1, S2 ... ..., Si locus amplifications product is detected, so as to obtain pair
Should be in the testing result of different sample of nucleic acid；
(3) testing result that step (2) is obtained is compared, so that it is determined that the genetic affinity between Different Individual.
In another preference, the genetic affinity analysis includes individual identification analysis, paternity test analysis, genetic connection
Analysis.
In another preference, described Different Individual comes from same family.
The features described above that the present invention is mentioned, or the feature that embodiment is mentioned can be in any combination.Disclosed in this case specification
All features can be used in combination with any combinations thing form, each feature disclosed in specification, can with it is any provide it is identical, equal
Deng or similar purpose alternative characteristics substitution.Therefore except there is special instruction, disclosed feature is only impartial or similar features
General example.
The present invention is advantageous in that：
(1) a kind of new str locus seat is provided, in the absence of CNV, and is not located at highly repetitive sequence region.
(2) heterozygosity of new str locus seat is high, high resolution.
(3) new str locus seat compatibility is high, can be used with existing most of sites in same system.
(4) new str locus seat or combined with known str locus seat, can effectively distinguish the genetic affinity of random crowd.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, it is no
Then percentage and number are percentage by weight and parts by weight.
Unless otherwise defined, all specialties used in text known to scientific words and one skilled in the art with anticipating
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the inventive method.Wen Zhong
Described preferable implementation only presents a demonstration with material to be used.
Embodiment 1
By the new conventional locus site of str locus seat G5S0003 and 10 of the present invention (VWA, D16S539, D5S818,
D7S820, D13S317, D8S1179, D18S51, TH01, D2S1338, AMELO) combination, using multiple fluorescence PCR check-up
Sample, 4 samples used are respectively akin family of three sample, and 1 affinity-less relation check sample.
G5S0003 and 10 conventional locus primer sequence is as shown in table 2.
The primer sequence of table 2
Note：Tetra- kinds of fluorescent dyes of PET, VIC, NED, FAM ((Applied Biosystems, USA companies).
Specific experiment operating procedure is as follows：
1) DNA sample prepares
Blood is gathered to the individual of sample of 4 selections；DNA sample is obtained by DNA extraction kit extracting and 1 μ is respectively taken
L, 1%agarose electrophoresis carry out quality examination and concentration sealing to its sample, then according to the concentration of estimation by Sample Dilution
To working concentration 5-10ng/ μ l.
2) PCR reacts
1ul samples are respectively taken, enter performing PCR reaction.
PCR reaction systems cumulative volume is 10 microlitres of (1 μ L10 × PCR buffer solutions (Qiagen companies), 0.2 μ L25mmol chlorine
Change magnesium, the mix primer of the multiple str locus seats of 1 μ L detections, 0.8 μ L2.5mmol dNTP, 0.04 μ L HotStarplus Taq
Enzyme (Qiagen companies), 5.96 μ L ddH2O).
PCR reaction conditions set as follows：95 DEG C 10 minutes；The Touchdown programs of 7 circulations (94 DEG C 20 seconds, 65 DEG C -1
DEG C/circulation 40 seconds and 72 DEG C of 2min), 28 cyclic amplifications (94 DEG C 20 seconds, 63 DEG C of 30 seconds and 72 DEG C of 2min), in 72 DEG C of extensions 2
Minute, extend at 60 DEG C 60 minutes, 4 DEG C of preservations；
After the completion of PCR reactions, pcr amplification product is diluted 20 times, 1ul is taken out and adds 8.9ul Hi-Di（Height deionization
Formamide）With 0.1ul LIZ（ABI house journals dyestuff, is used to mark molecule amount internal standard）, 95 DEG C of 5min, capillary after mixing
Electrophoresis tube loading；Because STR bit point detection primer used carries fluorescence, pass through capillary electrophoresis detection different colours fluorescence and difference
The am-plified fragments of size, STR parting information is drawn with fluorescence detecting system analysis result, as a result as shown in Fig. 1-4 and table 3.
The genotypic results of 11 locus are as shown in table 3；Sample 1 represents male parent, and sample 2 represents maternal, the table of sample 3
Show filial generation, sample 4 represents check sample.
The genotypic results of table 3
Note：X and Y represents sex chromosome, relative repetition that is digital then representing repeat unit in sample allele in form
Numerical value.
As a result show, sample 1 and sample 2, sample 3 can be substantially verified using obtained G5S0003 sites are newly screened
Between there is affiliation, with sample 4 exclude affiliation, newly screen obtained G5S0003 sites and shown in result
Higher discrimination, is applied in the biomedicine detection such as STR partings, paternity identification, individual identification, can effectively improve parting knowledge
Other efficiency and accuracy, and G5S0003 locus site has good compatibility, can be with most existing STR bases
Because seat point is applied in combination, there is very strong practicality.
Embodiment 2
20 groups of samples with affiliation and affinity-less relation are randomly selected, are detected using the method for embodiment 1,
Difference is, only with the new str locus seat G5S0003 of the present invention.
After testing, it is determined that after male parent sample, then determine to be left in 19 groups of samples with the akin filial generation sample of male parent
This, the new str locus seat G5S0003 of the present invention can accurately exclude the sample of wherein 11 groups affinity-less relations, show compared with
High discrimination.
Using the method for embodiment 1, by the new G5S0003 and 6 conventional locus site of str locus seat of the present invention
(VWA, D16S539, D18S51, TH01, D2S1338, AMELO) is combined, and remaining 8 groups of samples is detected, precise Identification
Its affiliation.
The genotypic results of 4 random 20 affinity-less relations of table and akin sample
Note：1. sample 1 represents male parent, and sample 2 represents maternal, and sample 3 represents filial generation, other sample affinity-less relations；②
To determine that sex adds AMELO；3. X and Y represent sex chromosome in form, digital then repeat unit in expression sample allele
Relatively heavy complex values.
As a result show, only pass through str locus of the present invention seat G5S0003, so that it may provide extremely valuable parting information.When
So, G5S0003 is combined with other known site can provide more accurately parting information, for example, additional three positions of G5S0003
Point D2S1338, D18S51 and D16S539, it is possible to accurately exclude the sample of affinity-less relation in remaining 8 groups, it is additional more
Site, the degree of accuracy is higher.It can be seen that in existing conventional Sites Combination, adding G5S0003, individual identification can be greatly improved
The degree of accuracy.
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (12)
1. a kind of tandem repeat loci G5S0003 of separation, it is characterised in that the sequence of the locus such as SEQ
ID NO.:Shown in 1.
2. a kind of nucleotide sequence of separation, it is characterised in that the structure of the nucleotide sequence is as follows：
X1-X2-X3
In formula, X1 is SEQ ID NO.:1-800 in 2；
X2 is the genetic polymorphism containing >=10 repeat unit atag and/or atga and/or taga and/or tata and/or gata
Area, and X2 length is 50-150bp, and genetic polymorphism area is SEQ ID NO.:Polynucleotides shown in 1, or lose
It is in SEQ ID NO. to pass polymorphic area:Polynucleotides formed by one or several repeat units are lacked or inserted on the basis of 1,
The repeat unit is atag and/or atga and/or taga and/or tata and/or gata；
X3 is SEQ ID NO.:925-1664 in 2.
3. tandem repeat loci G5S0003 as claimed in claim 1 purposes, it is characterised in that for preparing：
(a) it is used for reagent or kit that genetic affinity is analyzed；
(b) it is used for the reagent or kit of individual identification；
(c) it is used for the reagent or kit of paternity test or consanguinity analysis；
(d) it is used to detect reagent or kit with the presence or absence of female blood stains dye in extracting amniotic fluid；And/or
(e) be used to detecting leucocyte after bone-marrow transplantation in acceptor whether the kit replaced by donorcells.
4. purposes as claimed in claim 3, it is characterised in that described reagent includes (i) primer or primer pair；(ii) visit
Pin；(iii) nucleic acid chip.
5. purposes as claimed in claim 4, it is characterised in that the sequence of described primer pair such as SEQ ID NO.:3 and SEQ
ID NO.:Shown in 4.
6. the primer pair of a species-specific amplification tandem repeat loci G5S0003, it is characterised in that described primer
To sequence such as SEQ ID NO.:3 and SEQ ID NO.:Shown in 4, the sequence such as SEQ ID of the locus G5S0003
NO.:Shown in 1.
7. a kind of kit, it is characterised in that the kit includes：
(a) it is used for specific amplification tandem repeat loci G5S0003 primer pair, the sequence of the primer pair is such as
SEQ ID NO.:3 and SEQ ID NO.:Shown in 4, the sequence such as SEQ ID NO. of the locus G5S0003:Shown in 1；
(b) operation instructions.
8. kit as claimed in claim 7, it is characterised in that the kit is also selected from down including (c) specific amplification
The primer pair of one or more locus of group：
D3S1358、vWA、D16S539、D5S818、D7S820、CSF1PO、D13S317、TPOX、D8S1179、D21S11、
D18S51、TH01、FGA、D2S1338、D19S433、AMELO。
9. a kind of amplification system, it is characterised in that the amplification system includes：
(a) it is used for specific amplification tandem repeat loci G5S0003 primer pair, the sequence of the primer pair is such as
SEQ ID NO.:3 and SEQ ID NO.:Shown in 4, the sequence such as SEQ ID NO. of the locus G5S0003:Shown in 1；With
(b) primer pair of optional, one or more locus that specific amplification is selected from the group：
D3S1358、vWA、D16S539、D5S818、D7S820、CSF1PO、D13S317、TPOX、D8S1179、D21S11、
D18S51、TH01、FGA、D2S1338、D19S433、AMELO；
And the primer pair (a) and optional primer pair (b) are located at same amplification system.
10. amplification system as claimed in claim 9, it is characterised in that described amplification system is composite amplification system, its
In, described primer pair (b) includes 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, or 16 pairs of specific primers pair；
And/or
The composite amplification system is the composite amplification system of fluorescence labeling, wherein, the fluorescence labeling refers to from the following group
One or more fluorescent labeling reagents mark the primer of the locus：FAM, HEX, VIC, NED, PET, TAMRA, ROX, fluorescence
Element, FITC, IRD-700/800, CY3, CY5, CY3.5, CY5.5, TET, TAMRA, JOE, BODIPY TMR, Oregon are green, sieve
Red bright green, rhodamine is red, texas Red or Ya Jima are yellow.
11. a kind of amplification method, it is characterised in that including step：
In claim 9-10 in any described amplification system, using sample to be detected as template, polymerase chain reaction is utilized
The amplification of locus should be carried out, so as to obtain locus amplifications product.
12. a kind of genetic affinity analysis method, it is characterised in that including step：
(1), using sample to be detected as template, polymerase chain is utilized in any described amplification system in claim 9-10
Reaction carries out the amplification of locus, so as to obtain locus amplifications product；With
Wherein, it is >=2 that the sample to be detected, which includes i in the sample of nucleic acid S1, S2 ... ..., Si respectively from Different Individual, formula,
Positive integer；
(2) to from different sample of nucleic acid S1, S2 ... ..., Si locus amplifications product is detected, so as to be corresponded to
The testing result of different sample of nucleic acid；
(3) testing result that step (2) is obtained is compared, so that it is determined that the genetic affinity between Different Individual.
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| 人类Y染色体36个新STR位点的筛选与鉴定;彭冬铂 等;《遗传》;20121130;第34卷(第11期);1409-1416 * |
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