CN101956004A - Fluorescent-marked 16 X-STR loci multiplex amplification system and application thereof - Google Patents
Fluorescent-marked 16 X-STR loci multiplex amplification system and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioinstrumentation, in particular to a fluorescent-marked 16 X-STR loci multiplex amplification system and application thereof. The system performs multiplex amplification analysis on 16 X-STR loci, namely GATA165B12, DXS101, GATA172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, DXS6810 and DXS7132; the 16 loci are independent relatively and not linked mutually; and corresponding primers are respectively marked by four fluorescent markers, namely FAM, HEX, TAMRA and ROX. Based on the multiplex amplification system, an X-STR loci multiplex amplification kit suitable for Chinese people can be prepared for parentage analysis, individual recognition, sex tests, and gene location of X-linked inheritance disease, particularly for paternity identification of sisters, blooded half-sisters and skipped generations.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of detection system and application thereof of karyomit(e) somatotype, the fluorescence detecting system and the application thereof of particularly a kind of X chromosome str locus site somatotype.
Background technology
STR (short tandem repeats, be called for short STR) be by 2-7 base pair as core unit, a class microsatellite DNA sequence of connecting and repeating to form; STR is because the variation of core repeating unit number has formed the genetic polymorphism in str locus site.In the human genome, average every 6-10kb just has a str locus site, and the abundant source in high information gene site is provided for legal medical expert's individual identification and paternity test.
The allelic somatotype of STR has following outstanding feature in research and practical application: good repeatability, and the somatotype result is reliable and stable, and is simple to operate quick; Fragment length by pcr amplification, electropherotyping, is applicable to various samples and the detection of the sample of highly degrading easily generally at 100-400bp; Trinucleotide, tetranucleotide multiple STR site pcr amplification result are stable, and the additional band of generation, shadow band are few, easily somatotype; Be to draw the main tool that human inheritance figure, disease gene linkage analysis, genetic diseases diagnosis, individual recognition etc. are analyzed work.
X chromosome STR site extensively is present in the gene of eucaryote cell group, highly stable and have a higher genetic polymorphism, but compare with Y chromosome STR with euchromosome, the X chromosome STR site quantity of finding so far and using is considerably less, the information of aspects such as population distribution, mutation rate, linkage equilibrium, gene structure is still abundant inadequately, application in fields such as medical jurisprudence, medical science is limited, and is extensive far away from other DNA genetic marker.But X chromosome STR detects for paternity test, individual recognition, sex identification, X linkage inheritance Disease-causing gene location, tumor susceptibility gene, and there is special advantages aspects such as the molecular genetic mechanism research of the location of disease of multifactorial inheritance Disease-causing gene, morbidity, the design of medicine and use.So the relevant research of X chromosome has important meaning.
The research of X chromosome genetic marker also is in the initial stage at home, and commercialization detection kit in the world only has German Biotype Argus X-12 at present, and there is following problem in such test kit in the forensic application practice:
Meet difficulty when 1, adopting such test kit to increase genetic marker;
2, colony (the mainly white man colony) data that all is based on beyond the Chinese colony of these X-STR locus is developed, some locus wherein, and the gene frequency in Chinese colony distributes relatively poor, and the individual recognition ability is lower;
3, external commercial kit costs an arm and a leg.
These drawbacks limit the application at home of X chromosome genetic marker, be unfavorable for further promoting in basic unit.
Application number is 200810017404.6, and publication number is that the Chinese patent of CN101225387 discloses and a kind ofly covers 11 str locus seats of X chromosome total length and disclose the str locus seat carries out the karyomit(e) somatotype in conjunction with the allelotrope standard substance method.But the STR somatotype of this disclosure of the Invention is a silver dyes detection, though not high to the cost requirement of experimental installation, in experiment manual operation more, workload is big, experimental result is subject to the influence of personnel operation skill level.Application number is 200810219413.3, publication number is that the Chinese patent of CN101413030A discloses a kind of fluorescent composite amplification system that contains 10 X-STR locus, but 10 X-STR locus of this system are in same linkage group, need to calculate the haplotype frequency in practices such as paternity identification, whole discriminating usefulness reduces.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of can be fast in Chinese population, accurately at euchromosome STR, Y-STR be difficult to get rid of or assert the parental right relation special inspection case (as the father with female sister find relatives, the half-sister finds relatives, grandmother and granddaughter's evaluation etc.) the fluorescently-labeled X-STR locus composite amplification system identified.
Another object of the present invention be to provide above-mentioned composite amplification system be used for X linkage inheritance Disease-causing gene location, paternity test, with the father with female sister find relatives, field such as the half-sister finds relatives, grandmother and granddaughter's evaluation.
The present invention has set up a kind of fluorescently-labeled X-STR composite amplification system, this system can analyze 16 X-STR locus, and these 16 X-STR locus are: GATA165B12, DXS101, GATA172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, DXS6810 and DXS7132.
The present invention specifically comprises following content:
One, the preparation of allelic ladder.
Respectively to comprise 16 X chromosome str locus seat GATA165B12, DXS101, GATA172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, the not homoallelic plasmid of DXS6810 and DXS7132 is a template, be marked with the primer of fluorescence molecule to being primer with 5 ' end, the whole allelotrope in the above-mentioned X chromosome str locus of pcr amplification site, and the allelic clip size that goes out of definite pcr amplification, with whole allelic fragments according to etc. mixed in molar ratio obtain allelic ladder.
Two, the fluorescent mark of multiple PCR primer.
Owing to need carry out composite amplification to 16 X chromosome str locus seats, and to the restriction of equipotential gene fragment difference in length and amplicon length, will carry out the multi-fluorescence mark during multiplex PCR system construction, the present invention adopts 5 look fluorescent marks, considers following key element when selecting fluorescent mark:
1. the exciting light spectrum of every kind of fluorescein at interval should be even as far as possible, and it preferably is no less than 20nm at interval, to reach optimal separating efficiency;
2. the absorb light spectrum of a kind of exciting light spectrum of fluorescein and another kind of fluorescein is not overlapping as far as possible, to reduce mutual interference;
3. fluorescein can obtain by commercial sources, is not subjected to patent protection;
4. because following application mainly according to the genetic analyzer of American AB company, requires selected fluorescein to be suitable on the genetic analyzer of American AB company.
16 X-STR locus of the present invention adopt fluorescein-labelled (the 5th kind of fluorescence is used for interior target mark such as LIZ, SIZ etc.) of FAM, HEX, TAMRA and four kinds of different colours of ROX respectively.Be specially: FAM mark GATA165B12, DXS101, GATA172D05, HPRTB and DXS981 be totally 5 locus; HEX mark DXS8378, DXS6795, GATA31E08 and DXS6809 be totally 4 locus, and TAMRA mark DXS6803, DXS9902, DXS6807 and DXS7423 be totally 4 locus, and ROX mark DXS7133, DXS6810 and DXS7132 be totally three locus.
The inventor studies show that, can realize composite amplification in a PCR single tube, amplification efficiency height, no phase mutual interference.
Three, the composite amplification and the detection of 16 X-STR locus in the system of the present invention.
Composite amplification system of the present invention is to adopt composite PCR technology above-mentioned 16 the X-STR locus that increase simultaneously in same PCR reaction tubes, thus the chromosomal str locus seat of Analysis of X simultaneously.
Locus in the composite amplification system is positioned at a pair of primer amplification of these locus both sides, wherein 5 of the primer normal chain in the every pair of primer ' end is with fluorescein-labelled FAM, HEX, TAMRA and the ROX mark promptly used accordingly, and the primer of whole 16 X-STR locus proportionally mixes (in the test tube).
(1) PCR reaction system
The reaction system cumulative volume is 12-25 μ L, comprises PCR reaction buffer (Buffer) 1-2.5 μ L, primer mixture (Primer pair Mix) 1-3 μ L, and archaeal dna polymerase 1-2.5 U, template DNA 1-3 μ L, ultrapure water complements to cumulative volume.
(2) pcr amplification parameter
9700 or the PCR instrument of other models on increase, multiplex PCR amplification parameter is: 95 ℃ of 15min; 94 ℃ of 30sec, 57 ℃ of 90sec, 72 ℃ of 90sec carry out 30 circulations altogether; 60 ℃ are extended 60min.
Four, interpretation of result.
1. specimen preparation
Instantaneous centrifugal behind the mixing, in 96 orifice plates, every hole adds 10.0 μ L mixed solutions, adds PCR product 1 μ L again, and is of short duration centrifugal, and 95 ℃ of sex change 4 min place cooling 3 min in the ice chest, carries out electrophoresis with model genetic analyzers such as 3100 or 3130.
2. the setting of electrophoresis parameter
Process specifications according to genetic analyzer carries out writing of schedule of samples, and in schedule of samples, being provided with of electrophoresis parameter is as follows: Dye Set is E5, and Run Module selects " GeneScan36vb_POP4 Default ".
3. interpretation of result
Adopt GeneMapper ID v3.1 software or GeneMapper ID v3.2 or GeneMapper ID v3.3.
Method provided by the invention, be based on 16 str locus seat GATA165B12, DXS101, GATA172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, DXS6810 and DXS7132 of X chromosome, the all sites clip size is moderate, and PCR composite amplification and fluoroscopic examination all obtain satisfied result.
Fluorescently-labeled 16 str locus seat composite amplification systems of the present invention can the generate a reagent box, for fields such as medical jurisprudence paternity identification, individual recognition provide a kind of new detection system.
Above-mentioned composite amplification system can be applicable to special parental right relation evaluation or carries out individual recognition, sex identification and X linkage inheritance Disease-causing gene location etc.
Compared with prior art, the present invention has following beneficial effect:
1, highly sensitive: 16 str locus seat fluorescent composite amplification systems provided by the invention still can detect 16 whole str locus seats when the template amount is 25pg.
2,16 str locus seat fluorescent composite amplification systems provided by the present invention have been used for actual inspection case in this laboratory, and the result shows this system's polymorphism height, stability, good reproducibility, and the somatotype result is accurate, can satisfy actual needs.
3, the present invention at first develops one group of 16 X-STR locus is realized composite amplification in the single reaction pipe multicolored fluorescence labeling composite amplification detection system at home.
4, fluorescent dye primer composite amplification technology of the present invention is quick, easy, and the PCR product can be with genetic analyzer electrophoresis such as 310,3100,3130, and the result analyzes automatically, can stdn, can guarantee that the different experiments number of chambers is according to exactness relatively.
5, the present invention can prepare a cover test kit, carry out commercialization, can fill up the blank of the domestic X-STR of not having fluorescent composite amplification reagent kit, also can replenish the deficiency of present STR fluorescence detection reagent kit, as X-STR locus fluorescent composite amplification reagent kit with Chinese characteristics, can be used for paternity test, with the father with female sister find relatives, field such as the half-sister finds relatives, grandmother and granddaughter's evaluation, X linkage inheritance Disease-causing gene location.
Embodiment
The present invention is further elaborated below with reference to case study on implementation, and this enforcement is only used for illustrational purpose and limits the scope of the invention by any way by no means.
Embodiment 1Use 16 X-STR locus fluorescent composite amplification systems and carry out paternity test and individual recognition
Operation steps:
1, DNA extraction
The Chelex-100 method is extracted genomic dna: add the 1mL distilled water in the 1.5mL centrifuge tube, add 3 μ L whole bloods or 3 * 3mm blood cake then, mixing; Room temperature insulation 30min, interrupted oscillation; The centrifugal 5min of 14,000 r/min; Remove supernatant liquor.In precipitation, add 10% Chelex 100 (100mesh), 200 μ L; 56 ℃ of insulation 30min; 5-10Sec vibrates at a high speed; Boiling water bath 8min; The centrifugal 5min of 14,000 r/min gets supernatant liquor and is used for pcr amplification.
2, pcr amplification
The reaction cumulative volume is 25.0 μ L, comprises PCR reaction buffer (Buffer) 2.5 μ L, primer mixture (Primer pair Mix) 2.5 μ L, archaeal dna polymerase 0.5 μ L, template DNA 5.0 μ L (0.1ng/ μ L), ultrapure water 14.5 μ L.
In the above-mentioned primer mixture, the primer sequence and the fluorescent mark thereof of each locus are as shown in table 1.
16 X-STR locus of table 1 and primer sequence and fluorescent mark
| The X-STR locus | Primer sequence | Fluorescent mark |
| GATA165B12 | F: 5’-CTATGTATCATCAATCATCTATCCGTA-3’ | FAM |
| ? | R: 5’-GGTTGACTGTGATTCCTGGTTTA-3’ | ? |
| DXS101 | F: 5’-CTAAATCAGTCCAAATATCTCCCTT-3’ | FAM |
| ? | R: 5’-GTGCGCATGTATCCCAGAAC-3’ | ? |
| GATA172D05 | F:?5’-GGTTACCAGGGACTGGAGGA-3’ | ? |
| ? | R: 5’-AATAATTGAAAGCCCGGATTC-3’ | FAM |
| HPRTB | F: 5’-GGTGTCTCATGTAAGAGGGCAGT-3’ | ? |
| ? | R: 5’-ATTCAATAAATAGGAGAAGGGCAT-3’ | FAM |
| DXS981 | F: 5’-TTTCTTAGCTGGTCTGCCTTCTT-3’ | FAM |
| ? | R: 5’-GGATGAAAGAGTTGGAGATTGTG-3’ | ? |
| DXS8378 | F: 5’-GACAAGAACGAAACTCCAACTC-3’ | ? |
| ? | R: 5’-TTAGGCAACCCGGTGGTC-3’ | HEX |
| DXS6795 | F: 5’-GTGCTTGGTCACCTCAACATCA-3’ | ? |
| ? | R: 5’-TTCAGTGTTTGACATGGCTTTCT-3’ | HEX |
| GATA31E08 | F: 5’-AGATGTATAGACAGAGCTGGTGATG-3’ | HEX |
| ? | R: 5’-GTAACTTTATGGCAAGTTGAGGATC-3’ | ? |
| DXS6809 | F: 5’-TGCTTTAGGCTGATGTGAGGA-3’ | HEX |
| ? | R: 5’-GGCATGAAAAGAAGTTGGGTT-3’ | ? |
| DXS6803 | F: 5’-AAAGGAACATATGCTACTTTATCTCG-3’ | TAMRA |
| ? | R: 5’-GACCTAGAAATGTGCTTTGACAGG-3’ | ? |
| DXS9902 | F: 5’-GATGGAGTCTCTGGGTGAAGAG-3’ | ? |
| ? | R: 5’-CATATCAGGAGTATGGGATCACC-3’ | TAMRA |
| DXS6807 | F: 5’-GTCAGTTACTCATGGTAAAGTCCG-3’ | ? |
| ? | R: 5’-CAGTGGATGCCTGAAACCTC-3’ | TAMRA |
| DXS7423 | F: 5’-CATAGTAGGTGCCCAAAAGATATT-3’ | TAMRA |
| ? | R: 5’-GGATTCTACTGAGCATTTCGTG-3’ | ? |
| DXS7133 | F: 5’-AGGTGTAGCTTCCTTAGATGGC-3’ | ROX |
| ? | R: 5’-GTTCCAAGAATCAGAAGTCTCCTAC-3’ | ? |
| DXS6810 | F: 5’-GCCATACCCAGCCCTGAATA-3’ | ? |
| ? | R: 5’-CCTTTTGGGACCTTAAGTAAAATG-3’ | ROX |
| DXS7132 | F: 5’-CACTCCTGGTGCCAAACTCTA-3’ | ROX |
| ? | R: 5’-GTTGGTTCTTCTGATTCTGACTTTC-3’ | ? |
Above-mentioned 16 pairs of primers are designated as SEQ.ID.NO.1---SEQ.ID.NO.16 successively.
3, pcr amplification parameter: increase on 9700 PCR instrument, the pcr amplification parameter is: 95 ℃ of 15min; 94 ℃ of 30sec, 57 ℃ of 90sec, 72 ℃, 60sec carry out 30 circulations altogether; 60 ℃ are extended 60min.
4,3100 genetic analyzer electrophoresis: the process specifications according to genetic analyzer carries out writing of schedule of samples, and in schedule of samples, being provided with of electrophoresis parameter is as follows: Dye Set is E5, and Run Module selects " GeneScan36vb_POP4 Default ".
China Han, the Hui ethnic group, the Uygur nationality, the Mongols, the crowd's of Tibetan somatotype result has been investigated and obtained to 16 str locus seats of the present invention in population genetics in applicant's laboratory applications, prove that these genomic polymorphisms are higher, be suitable for the detection of Chinese population, and be applied in the actual inspection case and obtained ideal results.
SEQ.ID.NO.1 F:5’-CTATGTATCATCAATCATCTATCCGTA-3’
R:5’-GGTTGACTGTGATTCCTGGTTTA-3’
SEQ.ID.NO.2 F:5’-CTAAATCAGTCCAAATATCTCCCTT-3’
R:5’-GTGCGCATGTATCCCAGAAC-3’
SEQ.ID.NO.3 F:5’-GGTTACCAGGGACTGGAGGA-3’
R:5’-AATAATTGAAAGCCCGGATTC-3’
SEQ.ID.NO.4 F:5’-GGTGTCTCATGTAAGAGGGCAGT-3’
R:5’-ATTCAATAAATAGGAGAAGGGCAT-3’
SEQ.ID.NO.5 F:5’-TTTCTTAGCTGGTCTGCCTTCTT-3’
R:5’-GGATGAAAGAGTTGGAGATTGTG-3
SEQ.ID.NO.6 F:5’-GACAAGAACGAAACTCCAACTC-3’
R:5’-TTAGGCAACCCGGTGGTC-3’
SEQ.ID.NO.7 F:5’-GTGCTTGGTCACCTCAACATCA-3’
R:5’-TTCAGTGTTTGACATGGCTTTCT-3’
SEQ.ID.NO.8 F:5’-AGATGTATAGACAGAGCTGGTGATG-3’
R:5’-GTAACTTTATGGCAAGTTGAGGATC-3’
SEQ.ID.NO.9 F:5’-TGCTTTAGGCTGATGTGAGGA-3’
R:5’-GGCATGAAAAGAAGTTGGGTT-3’
SEQ.ID.NO.10 F:5’-AAAGGAACATATGCTACTTTATCTCG-3’
R:5’-GACCTAGAAATGTGCTTTGACAGG-3’
SEQ.ID.NO.11 F:5’-GATGGAGTCTCTGGGTGAAGAG-3’
R:5’-CATATCAGGAGTATGGGATCACC-3’
SEQ.ID.NO.12 F:5’-GTCAGTTACTCATGGTAAAGTCCG-3’
R:5’-CAGTGGATGCCTGAAACCTC-3’
SEQ.ID.NO.13 F:5’-CATAGTAGGTGCCCAAAAGATATT-3’
R:5’-GGATTCTACTGAGCATTTCGTG-3’
SEQ.ID.NO.14 F:5’-AGGTGTAGCTTCCTTAGATGGC-3’
R:5’-GGATTCTACTGAGCATTTCGTG-3’
SEQ.ID.NO.15 F:5’-GCCATACCCAGCCCTGAATA-3’
R:5’-CCTTTTGGGACCTTAAGTAAAATG-3’
SEQ.ID.NO.16 F:5’-CACTCCTGGTGCCAAACTCTA-3’
R:5’-GTTGGTTCTTCTGATTCTGACTTTC-3’
Claims (7)
1. one kind fluorescently-labeled 16 heavy X-STR locus composite amplification system is characterized in that 16 locus of this composite amplification system are as follows: GATA165B12, DXS101, GATA172D05, HPRTB, DXS981, DXS8378, DXS6795, GATA31E08, DXS6809, DXS6803, DXS9902, DXS6807, DXS7423, DXS7133, DXS6810, DXS7132.
2. according to the described fluorescently-labeled X-STR locus composite amplification system of claim 1, it is characterized in that described 16 locus carry out pcr amplification simultaneously in a composite amplification system.
3. according to the described fluorescently-labeled X-STR locus composite amplification system of claim 1, it is characterized in that described 16 locus primers use FAM, HEX, four kinds of TAMRA, ROX fluorescein-labelled respectively.
4. according to claim 3 described fluorescently-labeled 16 heavy X-STR locus composite amplification systems, it is characterized in that four kinds of fluorescein-labelled 16 locus primers, specific as follows: FAM mark GATA165B12, DXS101, GATA172D05, HPRTB and DXS981 be totally 5 locus; HEX mark DXS8378, DXS6795, GATA31E08 and DXS6809 be totally 4 locus, and TAMRA mark DXS6803, DXS9902, DXS6807 and DXS7423 be totally 4 locus, and ROX mark DXS7133, DXS6810 and DXS7132 be totally three locus.
5. according to claim 1,2,3 or 4 described fluorescently-labeled 16 heavy X-STR locus composite amplification systems, it is characterized in that 16 X-STR locus and primer sequence and fluorescein-labelled thereof, concrete corresponding as follows:
。
6. test kit based on the described fluorescently-labeled 16 heavy X-STR locus composite amplification systems of one of claim 1-4, it is characterized in that comprising: the PCR reaction system, the reaction system cumulative volume is 12-25 μ L, comprise PCR reaction buffer 1-2.5 μ L, primer mixture 1-3 μ L, archaeal dna polymerase 1-2.5 U, template DNA 1-3 μ L, all the other are ultrapure water;
Described primer mixture comprises: 16 X-STR locus and primer sequence and fluorescein-labelled thereof, concrete corresponding as follows:
。
7. one of claim 1 to 4 described fluorescently-labeled 16 heavy X-STR locus composite amplification systems are used in special parental right concerns the assignment of genes gene mapping of evaluation or individual recognition, sex identification and X linkage inheritance disease.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108179199A (en) * | 2018-02-28 | 2018-06-19 | 公安部物证鉴定中心 | Kit and its primer special combination based on two generation sequencing technologies detection X-STR locus |
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| CN108676856A (en) * | 2018-04-17 | 2018-10-19 | 深圳华大法医科技有限公司 | Mankind's X chromosome STR bit point detecting system, DNA sample analysis method and primer pair |
| CN110894531A (en) * | 2018-09-12 | 2020-03-20 | 深圳华大法医科技有限公司 | STR locus set for pig and application |
| CN112195228A (en) * | 2020-09-28 | 2021-01-08 | 苏州阅微基因技术有限公司 | X-STR fluorescent amplification system, kit and application |
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| CN114410798A (en) * | 2021-12-20 | 2022-04-29 | 中山大学 | Multiplex amplification detection system for chain STR loci on human chromosome I and chromosome II and application thereof |
| CN114410798B (en) * | 2021-12-20 | 2023-11-03 | 中山大学 | Composite amplification detection system of linked STR loci on human chromosomes 1 and 2 and its application |
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