CN1377973A - Sex chromosome short tandem repetitive sequence four site complex amplificatino kit and amplification method - Google Patents
Sex chromosome short tandem repetitive sequence four site complex amplificatino kit and amplification method Download PDFInfo
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- CN1377973A CN1377973A CN 01111085 CN01111085A CN1377973A CN 1377973 A CN1377973 A CN 1377973A CN 01111085 CN01111085 CN 01111085 CN 01111085 A CN01111085 A CN 01111085A CN 1377973 A CN1377973 A CN 1377973A
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Abstract
The present invention provides X and Y sex chromosome short tandem repetitive sequence four site complex amplification kit capable of typing STR allelic genes of four X and Y sex chromosome sites. The main components in the kit include primer pairs HPRT, DXS6799, DXS6804 and DXS7132 and/or primer pairs DYS389, DY390, DYS19 and DYS388, auxiliary components KCl 50A mol/L, Tris-HCl (pH8.4)10A mmol/L, MgC12 1.5A mmol/L, glutin 0.05A g/L and nDTP:0.15 A mmol/L. The present invention has the advantages of capacity of simultaneous detection of four STR sits of X or Y sex chromosome and high individual distinguishing capacity in every experiment.
Description
The present invention relates to molecular biology, medical jurisprudence, genetics field polymerase chain reaction composite amplification technology.
STR (short tandem repeat, STR) be a class tumor-necrosis factor glycoproteins in the human genome, for the STR on certain locus (claiming the site again), its repeating unit (claiming core sequence again) is identical often, in the crowd between the Different Individual because the multiplicity difference of core sequence, this segmental DNA length is also different.Various STR fragment (allelotrope) on the same locus, that exist between the Different Individual can be increased and shows with amplification technique.The STR sequence is distributed widely in the chromosomal dna sequence dna.A large amount of genetics mass surveies prove that the STR fragment (allelotrope) in euchromosome is relayed according to a mendelian inheritance generation generation.
X, Y chromosome are the sex chromosome of human body.X chromosome is passed to children by mother, and Y chromosome is then passed to son by father.In sex chromosome, have many STR site equally, and these sites are to transmit in the mode of haplotype.Therefore, can come discriminate individuals by the haplotype that the allelotrope that detects the STR site on X or the Y chromosome is respectively formed, or determine whether there is one's own relation between controversial mothers and sons, mother and daughter or the father and son.But, because the allelotrope limited amount that single STR site is contained, so resolving power is not high.
In order to improve the resolving power that detects the STR site, way in the past is to increase the STR site of detecting.But experiment can only increase a site each time.Like this, increased workload greatly, time and effort consuming.And in detecting practice, the amount of delivering to breadboard sample often seldom is not enough to do repeatedly experiment and uses.How to address this problem very important.
The purpose of this invention is to provide a kind of new test kit, this test kit can be simultaneously to the STR allelic gene typing in X or heterosomal four sites of Y, and resolving power is obviously improved.
Second purpose of the present invention provides a kind of new amplification method, to improve specificity.
Test kit master composition of the present invention is
The X primer is to HPRT, DXS6799, DXS6804 and DXS7132, or/and the Y primer is to DYS389, DYS390, DYS19 and DYS388.
The right concentration of each primer is 0.1～0.4A μ mol/L, wherein A=1～50.
The auxiliary composition of test kit of the present invention is
Reaction buffer composition: include KCl 50Ammol/L, Tris-HCl (pH8.4) 10Ammol/L, MgCl
21.5Ammol/L, gelatin 0.05Ag/L, wherein A=1～50.
DNTP:0.15Ammol/L, wherein A=1～50.
Test kit of the present invention also can comprise
Taq archaeal dna polymerase 0.5 ~ 1A U, wherein A=1～50.
Above-mentioned A value can be greater than 50, promptly all ingredients can be mixed with denseer, or to saturated or solid.Dilute with water during use.
Above reagent is the composition that uses when simultaneously the STR allelic gene typing in X or heterosomal four sites of Y being carried out pcr amplification reaction.They are put together, make test kit, use with convenient.When using test kit of the present invention and carrying out pcr amplification reaction, it is made into the PCR reaction solution, adds micro-tested template DNA, carry out thermal cycling by the amplification method of introducing below.
Test kit of the present invention can not contain the Taq archaeal dna polymerase, and adds simultaneously in when reaction and tested template.
Amplification reaction method of the present invention comprises the steps:
1, above-mentioned primer, reaction buffer, dNTP, TaqDNA polysaccharase and tested template DNA are mixed, in case of necessity thin up;
2, mixed reaction solution is carried out thermal cycling: 95 ℃ of 5min; 94～95 ℃ of 45S then, 50-60 ℃ of 45S, 70-72 ℃ of 1min, 30 circulations; Last 72 ℃ of 5min.
Provide embodiment below.One, test kit: comprise
The X primer is to HPRT, DXS6799, DXS6804 and DXS7132, or/and the Y primer is to DYS389, DYS390, DYS19 and DYS388, the right concentration of each primer is 1 μ mol/L;
Reaction buffer composition: include KCl 500mmol/L, Tris-HCl (pH8.4) 100mmol/L, MgCl
215mmol/L, gelatin 0.5g/L;
dNTP：1.5mmol/L；
Taq archaeal dna polymerase 5U.Two, PCR reaction content preparation
With above-mentioned test kit (A=10) reagent reactant ligand operation liquid, cumulative volume 50 μ l.Contain KCl 50mmol/L, Tris-HCl (pH8.3) 10mmol/L, MgCl
21.5mmol/L, gelatin 0.05 ~ 0.15g/L, dNTP 0.15mmol/L, X or Y primer are to each 0.1 ~ 0.4 μ mol/L, and Taq archaeal dna polymerase 0.5 ~ 1U adds tested template DNA 0.5 ~ 20ng.Three, thermal cycling
Pre-95 ℃ of 5min of sex change; 94 ℃ of 45s then, 50～60 ℃ of 45s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min again.4 ℃ of preservations.Four, conventional electrophoretic analysis
Concentration be 6% (Acr: Bis=19: polyacrylamide gel 1), gel slab is 18 * 30cm greatly, thick 0.5mm, electrode buffer are 0.5 * TBE (going up the utmost point), 3mol/L NaAc/1 * TBE (=1: 2) (the following utmost point).2 μ l amplified productions add sample damping fluid mixing, with allelic ladder electrophoresis side by side, permanent power 25W, electrophoresis 2hrs.Silver dyes colour developing.
10% Glacial acetic acid is 20min fixedly, and ultrapure washing 3 times is with 12mmol/L cma staining 30min, ultrapure washing 10s places staining fluid (37% formaldehyde 0.7ml, anhydrous sodium carbonate 30g, add water to 1L) in dye clearly to band, 10% Glacial acetic acid is ended colour developing, dried glue is preserved.
Good effect of the present invention and advantage:
Because the frequency distribution of the haplotype that the several STR site that is chained together forms is well below single The gene frequency in STR site, so its individual resolution capability improves greatly. In general, the allelic amplified fragments of each of STR site is all shorter, when sample part corruption makes DNA wherein is during Partial digestion, still might amplify the STR site by round pcr Allele. Now, utilize composite amplification reagent kit, can detect simultaneously X or Y chromosome at every turn 4 STR sites, making once, experiment can reach very high individual identification power. Reality at legal medical material evidence examination In trampling, often the amount of running into sample few and that part is corrupt need to carry out dna typing and makes individual identification, the spy Be not in some gang-rape cases, in the time of determining the suspicion object, it is very heavy that the Y chromosome dna typing just seems Want and effectively. Carrying out some special Cases of Disputed Parentages (as will be to controversial mothers and sons, mother and daughter, father and son Determine whether one's own relation) evaluation the time, the multidigit point composite amplification somatotype examination that X, Y chromosome are special The agent box also has special value, can shorten the time, and the saving funds lighten one's labor, and raise the efficiency. Cause This, the present invention will produce good society and economic benefit. 1. because this method is easy, for setting up the relevant dna fingerprint database of large-scale crowd, provide fine Technical guarantee. 2. in the situation of many particular difficulty, part is putrid and deteriorated (such as the injured party when longer again less such as sample Between just be found) time, still might scout to solve a case for public security organs provides scientific evidence. 3. kit can be some hereditary disease or Genetic predisposition to disease (particularly sex-linked genetic disease) The dna fragmentation correlation analysis provides instrument. 4. can be the anthropological studies gives information. 5. simple, economy is easily promoted, and good society and economic benefit are arranged.
Claims (4)
1, a kind of sex chromosome short tandem repetitive sequence four site composite amplification reagent kit is characterized in that: main composition X primer is to HPRT, DXS6799, DXS6804 and DXS7132, or/and the Y primer is to DYS389, DYS390, DYS19 and DYS388; The right concentration of each primer is 0.1～0.4A μ mol/L, wherein A=1～50.
2, sex chromosome short tandem repetitive sequence four site composite amplification reagent kit according to claim 1 is characterized in that also comprising:
Reaction buffer composition: include KCl 50Ammol/L, Tris-HCl (pH8.4) 10Ammol/L, MgCl
21.5Ammol/L, gelatin 0.05Ag/L, wherein A=1～50;
DNTP:0.15Ammol/L, wherein A=1～50.
3, sex chromosome short tandem repetitive sequence four site composite amplification reagent kit according to claim 1 is characterized in that also can comprising: Taq archaeal dna polymerase 0.5 ~ 1A U, wherein A=1～50.
4, a kind of TRAP is characterized in that comprising the steps:
(1), with primer, reaction buffer, dNTP, TaqDNA polysaccharase and the mixing of tested template DNA, thin up in case of necessity;
(2), mixed reaction solution is carried out thermal cycling: 95 ℃ of 5min; 94～95 ℃ of 45S then, 50-60 ℃ of 45S, 70-72 ℃ of 1min, 30 circulations; Last 72 ℃ of 5min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01111085 CN1377973A (en) | 2001-04-02 | 2001-04-02 | Sex chromosome short tandem repetitive sequence four site complex amplificatino kit and amplification method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01111085 CN1377973A (en) | 2001-04-02 | 2001-04-02 | Sex chromosome short tandem repetitive sequence four site complex amplificatino kit and amplification method |
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| CN1377973A true CN1377973A (en) | 2002-11-06 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323877B (en) * | 2007-06-15 | 2011-05-11 | 中山大学达安基因股份有限公司 | Reagent box for detecting No 21 chromosome and idiochromosome number abnormality |
| CN107122625A (en) * | 2016-02-24 | 2017-09-01 | 北京爱普益生物科技有限公司 | The processing method of mankind's Short tandem repeats sequence high-flux sequence information |
-
2001
- 2001-04-02 CN CN 01111085 patent/CN1377973A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323877B (en) * | 2007-06-15 | 2011-05-11 | 中山大学达安基因股份有限公司 | Reagent box for detecting No 21 chromosome and idiochromosome number abnormality |
| CN107122625A (en) * | 2016-02-24 | 2017-09-01 | 北京爱普益生物科技有限公司 | The processing method of mankind's Short tandem repeats sequence high-flux sequence information |
| CN107122625B (en) * | 2016-02-24 | 2020-10-09 | 北京爱普益生物科技有限公司 | Method for processing high-throughput sequencing information of human short segment tandem repeat sequence |
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