CN101608233A - The fluorescence detection method of a kind of X chromosome str locus site somatotype - Google Patents
The fluorescence detection method of a kind of X chromosome str locus site somatotype Download PDFInfo
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- CN101608233A CN101608233A CNA2009100233975A CN200910023397A CN101608233A CN 101608233 A CN101608233 A CN 101608233A CN A2009100233975 A CNA2009100233975 A CN A2009100233975A CN 200910023397 A CN200910023397 A CN 200910023397A CN 101608233 A CN101608233 A CN 101608233A
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Abstract
The invention discloses the fluorescence detection method of a kind of X chromosome str locus site somatotype, comprise the preparation of allelic ladder: the plasmid with the different genotype that comprises 3 X chromosome str locus site DXS7132, DXS6799 and DXS6804 is a template respectively, the primer that is marked with fluorescence molecule with 5 ' end mixes whole allelic fragments obtaining allelic ladder to being the primer PCR amplification according to the molecular weight geometric ratio; The STR allelotrope of 3 X chromosomes that comprise at same amplification system pcr amplification X chromosome to be measured; The fluoroscopic examination somatotype with pcr amplification to the allelic ladder that has the fluorescence molecule mark and the STR allelotrope fragment of X chromosome to be measured carry out capillary electrophoresis separation and carry out the stdn somatotype.The present invention is applied to detecting of paternity test, individual recognition, sex identification, X linkage inheritance Disease-causing gene location, tumor susceptibility gene.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of detection method of karyomit(e) somatotype, the fluorescence detection method 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 significant advantage 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 important tool that human inheritance figure, disease gene linkage analysis, genetic diseases diagnosis, individual recognition etc. are analyzed work.
The somatotype of STR, at present the most frequently used is to use pcr amplification STR, amplified production is carried out polyacrylamide gel electrophoresis, determine genotype and calculate gene frequency and genetic distance and constructing system growth tree according to segmental size, dye in conjunction with silver for available autography technology of the detection of PCR product or PAGE.
X chromosome STR site extensively exists 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 markers.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-8 at present, and there is following problem in such test kit in the forensic application practice:
Meet difficulty when 1) adopting this class 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 gene locuss is developed, some gene locus wherein, and it is relatively poor to distribute in the gene frequency of Chinese colony, and individual recognition capability 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 a kind of 11 str locus sites that cover the X chromosome total length; And the karyomit(e) somatotype is carried out in the str locus site in conjunction with the allelotrope standard substance method disclosed.But the STR somatotype of this disclosure of the Invention is a silver to be dyed, and silver dyes detection, not high to the cost requirement of experimental installation, and test operation simply is easy to grasp, but manual operation is more in the experiment, and workload is big, and experimental result is influenced by personnel's skill level.
Summary of the invention
Technical problem to be solved by this invention is to provide the fluorescence detection method of a kind of X chromosome str locus site somatotype, this method has bigger using value stdn somatotype by the fluorescence somatotype to 3 X chromosome STR sites in medicolegal individual's identification and paternity test
The present invention is achieved through the following technical solutions:
The fluorescence detection method of a kind of X chromosome str locus site somatotype may further comprise the steps:
1) preparation of allelic ladder:
Plasmid with the different genotype that comprises 3 X chromosome str locus site DXS7132, DXS6799 and DXS6804 is a template respectively, 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;
Wherein, the primer of DXS7132 gene locus pcr amplification is to being:
Upstream primer: agcccatttt cataataaat cc 22;
Downstream primer: fluorescence molecule-aatcagtgct ttctgtacta ttgg 24;
The primer of DXS6799 gene locus pcr amplification is to being:
Upstream primer: fluorescence molecule-atgaattcag aattatcctc atacc 25;
Downstream primer: gaaccaacct gcttttctga 20;
The primer of DXS6804 gene locus pcr amplification is to being:
Upstream primer: fluorescence molecule-cccagatatt ttgaccacca 20;
Downstream primer: ggcatgtggt tgctataacc 20;
2) the STR allelotrope of pcr amplification X chromosome to be measured:
With the genome that comprises X chromosome to be measured is template, with the primer of the str locus site pcr amplification of described 3 X chromosomes of step 1) to being primer, the STR allelotrope of 3 X chromosomes that comprise at same amplification system pcr amplification X chromosome to be measured;
3) fluoroscopic examination somatotype
With pcr amplification to the allelic ladder that has the fluorescence molecule mark and the STR allelotrope fragment of X chromosome to be measured carry out capillary electrophoresis separation; Adopt the fluorescence developing method, X chromosome to be measured is carried out the stdn somatotype according to the electrophoretic separation result.
Described fluorescence molecule is FAM, JOE, ROX, HEX or TMR.
Described pcr amplification parameter is: 94 ℃ of pre-sex change 6min, and 94 ℃ of sex change 30 seconds, 60 ℃ of renaturation 35 seconds, 72 ℃ were extended 30 seconds, 30 circulations, last 72 ℃ are extended 10min.
The tumor-necrosis factor glycoproteins of described DXS7132 gene locus is cata, comprises 6 allelotrope, and the allelotrope clip size of pcr amplification is 278-298bp.
The tumor-necrosis factor glycoproteins of described DXS6799 gene locus is tatc, comprises 8 allelotrope, and the allelotrope clip size of pcr amplification is 230-258bp.
The tumor-necrosis factor glycoproteins of described DXS6804 gene locus is atag, comprises 10 allelotrope, and the allelotrope clip size of pcr amplification is 161-197bp.
Compared with prior art, the present invention has following beneficial technical effects:
Method provided by the invention, 3 str locus site DXS7132, the DXS6799 and the DXS6804 that are based on X chromosome unite identification, and wherein, pcr amplified fragment length is respectively DX6804 site: 161-197bp; DXS6799 site: 230-258bp; DXS7132 site: 278-298bp, all sites clip size is moderate, and PCR composite amplification and fluoroscopic examination all obtain satisfied result.
Above-mentioned 3 X chromosome str locus loci detection methods are used for Chinese nation's population genetic data investigation, and obtained the somatotype result of a plurality of Ethnic Populations, learn by statistics and handle proof, these 3 X-STR sites can be applied in the aspects such as individual recognition, paternity test and gene diagnosis in fields such as medical jurisprudence, anthropology, genetics and disease fully, the result shows that population of China and external crowd X-STR genetics characteristics have significant difference, are with Chinese characteristics.
DXS6804 gene locus individual recognition power is greater than 0.3220 in Chinese population; DXS6799 gene locus individual recognition power is greater than 0.1286; DXS7132 gene locus individual recognition power is greater than 0.6801; And by after 3 X-STR site DXS6804, DXS6799, the DXS7132 associating, individual recognition rate result is greater than 0.9514, and being presented in medicolegal individual's identification and the paternity identification has bigger using value.
The present invention has adopted composite amplification, is about to manyly primer is put into same amplification system increase according to a compromise condition; The result shows, DXS6804, DXS6799, it is feasible that the method for DXS7132 gene locus fluorescent dye primer composite amplification detects on the capillary electrophoresis automatic analysis system, carries out composite amplification checking native system by the sample that uses the known type and has somatotype effect accurately.
With in the classifying method of existing STR the developing technique of pcr amplification product or silver are dyed technique of display and compare, the invention provides the fluorescence detection method of the somatotype of STR, fluorescence molecule is marked on the primer, pass through the fluorescence technique of display again to the allelotrope fragment somatotype after the electrophoretic separation, fluorescent dye primer detects by automatic sequencer, and level of automation height, instrument detect somatotype automatically, objective and accurate, the good reproducibility of result is convenient to realize normalizing operation; Wherein the making of the optimization of PCR primer and amplification condition and somatotype marker makes the present invention can accomplish that stdn and simplification and suitable grass-roots unit are universal.
The present invention has overcome the defective of using external test kit can't reflect Chinese multi-national genetic characteristics, can be applied to detecting of paternity test, individual recognition, sex identification, X linkage inheritance Disease-causing gene location, tumor susceptibility gene, 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 are with a wide range of applications.
Description of drawings
Fig. 1 is the plasmid map of pGEM-T Easy Vector carrier;
Fig. 2 is that the electrophoresis fluorescence of allelotrope standard substance detects the somatotype result;
Fig. 3 is that the electrophoresis fluorescence of sample 1 detects the somatotype result;
Fig. 4 is that the electrophoresis fluorescence of sample 2 detects the somatotype result;
Fig. 5 is that the electrophoresis fluorescence of sample 3 detects the somatotype result.
Embodiment
Below in conjunction with accompanying drawing the present invention is elaborated, the explanation of the invention is not limited.
The invention provides a kind of fluorescence classifying method of uniting identification, the tumor-necrosis factor glycoproteins in above-mentioned str locus site, allelotrope number (repeat number), clip size and as shown in table 1 at the particular content of X chromosome position based on 3 str locus site DXS7132, DXS6799 and the DXS6804 of X chromosome.
Table 1 gene locus information
| The site | Tumor-necrosis factor glycoproteins | The allelotrope number | Clip size (bp) | Chromosome position (Mb) |
| ??DXS6799 | ??tatc | ??8 | ??230-258 | ??92.65 |
| ??DXS6804 | ??atag | ??10 | ??161-197 | ??110.8 |
| ??DXS7132 | ??cata | ??6 | ??278-298 | ??60.8 |
Described fluorescence classifying method is to hold the mark fluorescent molecule at 5 ' of its primer when the STR site of allelic standard substance in the STR site of pcr amplification or X chromosome to be measured, shows by fluorescence to replace existing silver to dye; Determine its somatotype result by the STR site of X chromosome to be measured and the contrast of allelic standard substance.
At first design the primer of STR site pcr amplification in the aforesaid method, and at 5 ' end mark fluorescent molecule.3 str locus site upstream and downstream primer sequences of X chromosome see Table 2, the mark of fluorescence molecule is finished by commercial test kit, the molecule of institute's mark can be FAM, JOE, ROX, HEX or TMR, the dna molecular of the known length that indicates the red fluorescence molecule of the error of its colouring discrimination when being used to get rid of each capillary electrophoresis as interior target.
The instrument that is used for the fluoroscopic examination of STR allelotrope has many kinds.The present invention considers and detects reliably that price moderate selected FAM as fluorescent marker in the specific implementation; Corresponding fluoroscopic examination is for discerning the indicating system of FAM fluorescence, and wherein, the ABI377 genetic analyzer is comparatively universal, and other genetic analyzer that can discern FAM fluorescence also can be used.
Table 2 has 3 X chromosome STR site primers of FAM mark to sequence
| The site | Upstream primer | Downstream primer |
| ??DXS6799 | ??FAM-atgaattcag?aattatcctc?atacc | ??gaaccaacct?gcttttctga |
| ??DXS6804 | ??FAM-cccagatatt?ttgaccacca | ??ggcatgtggt?tgctataacc |
| ??DXS7132 | ??agcccatttt?cataataaat?cc | ??FAM-aatcagtgct?ttctgtacta?ttgg |
Detailed detection method of the present invention is:
1) preparation of allelic standard substance (ladder):
A, comprise the preparation of str locus site different genotype plasmid:
Adopt clone's test kit, the PCR product that will have the homozygous STR site of different genotype (core sequence multiplicity difference) is connected with plasmid vector respectively, the present invention specifically with pGEM-T EasyVector carrier (U.S. Promage company) as carrier is carrier, its plasmid map as shown in Figure 1, the connection site in polyclone zone is between EcoRI and the SpeI enzyme point of contact;
Plasmid transformed competence colibacillus cell after b, the reorganization is selected to cultivate according to blue hickie, behind the extraction plasmid, filters out with the pcr amplification method and contains the segmental positive colony of correct insertion.Adopt the full-automatic genetic analyzer of ABI3730, check order, determine to insert segmental size and composition with the insertion fragment of the public M13 forward primer of plasmid to recombinant plasmid; The correct allele that contains that confirms through order-checking is inserted segmental plasmid and carries out enlarged culturing, and behind the bacterium liquid glycerol adding that obtains ,-70 ℃ of preservations are standby;
C, to insert segmental recombinant plasmid dna be template to contain allelotrope, 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, compare with 25bp DNA marker, order-checking sample and standard cell lines strain GM9947A simultaneously, determine the allelic clip size that pcr amplification goes out; Wherein, the DXS7132 gene locus comprises that 6 allelotrope, DXS6799 gene locus comprise 8 allelotrope, the DXS6804 gene locus comprises 10 allelotrope;
The parameter of pcr amplification is: 94 ℃ of pre-sex change 6min, and 94 ℃ of sex change 30 seconds, 60 ℃ of renaturation 35 seconds, 72 ℃ were extended 30 seconds, 30 circulations, last 72 ℃ are extended 10min;
D, with whole allelic fragments according to etc. mixed in molar ratio, make the different lengths dna segment that contains equal amts in the mixed solution, even the allelic fragment molecular amounts that every kind amplifies in the allelic ladder reaches 1: 1 state.
Be prepared into the allelic ladder of three locus, and, verify the operability of gained allelic ladder by the method that silver dyes colour developing and ABI 377 genetic analysis instrument automatic parting directions.
2) the STR allelotrope of PCR composite amplification X chromosome to be measured
A, be template, adopt and the allelotrope mark identical primer that increases, the upstream and downstream primer is diluted to 100 μ mol/L,, take out 10 μ l and be diluted to 5 μ mol/L, as working fluid as mother liquor with the genome that comprises X chromosome to be measured; The volume of pcr amplification system is 12.5 μ L, contains 1 * reaction buffer, 1.5mmol/L Mgcl2, the 1UTaq enzyme, 0.12pmol/ μ L DXS6804 primer, 0.2pmol/ μ LDXS6799 primer, 0.2pmol/ μ L DXS7132 primer, 200 μ mol/L dNTP, DNA20～200ng;
The pcr amplification underlying parameter is: 94 ℃ of pre-sex change 6min, and 94 ℃ of sex change 30 seconds, 60 ℃ of renaturation were extended 30 seconds for 35 seconds, 72 ℃, amounted to 30 circulations, and last 72 ℃ are extended 10min.
3) fluoroscopic examination somatotype
Use ABI377 genetic analyzer (U.S. PE company) to pcr amplification to the allelic ladder that has the fluorescence molecule mark and the STR allelotrope fragment of X chromosome to be measured carry out capillary electrophoresis separation; Because primer has indicated fluorescence molecule, the pcr amplification after product has promptly had fluorescence molecule, in genetic analyzer during electrophoresis, instrument can be discerned the optical signal that fluorescence molecule sends, and be treated to data and show signal via computer, adopt the fluorescence developing method X chromosome to be measured to be carried out the stdn somatotype according to the electrophoretic separation result:
Referring to Fig. 2-Fig. 5, the electrophoresis fluorescence of allelotrope standard substance detects the somatotype result as shown in Figure 2, and the electrophoresis fluorescence that Fig. 3,4,5 is respectively sample 1,2,3 detects the somatotype result; Wherein, from left to right be followed successively by the somatotype result of DXS6804, DXS6799, DXS7132 gene locus, the allelotrope standard substance has comprised whole allelotrope of 3 gene locuss, and sample 1 and sample 2 are the male sex, have only an X chromosome, genotype is respectively 13/11/14; 12/12/14 is unimodal (allelotrope in corresponding str locus site name on the described digitized representation X chromosome); Sample 3 is the women, because there are two X chromosomes, genotype is (12,14)/(12,12)/(13,13), show as in the DXS6804 site bimodal, other two sites because allelotrope identical, show as unimodal; Though there is identical genotype in single site in above-mentioned three samples, three gene locuss are united, and can draw the conclusion that three samples derive from Different Individual.
According to this classifying method, just can be applied to fields such as medical jurisprudence, solve practical problemss such as individual recognition, paternity test.And amplified production uses composite amplification method associating fluorescent mark classifying method to detect somatotype, and required instrument and equipment is simple, and is easy to operate, and detected result can stdn, can be directly used between the laboratory resource sharing and set up the genetic resources database.The present invention has filled up the domestic blank that does not have standardized X-STR detection technique and scheme, and obtains satisfied result in a plurality of nationalitys of China; Utilize the X chromosome STR parting kit of this technology preparation, can commercially produce and be extended to domestic each laboratory and use.
Claims (6)
1, the fluorescence detection method of a kind of X chromosome str locus site somatotype is characterized in that, may further comprise the steps:
1) preparation of allelic ladder:
Plasmid with the different genotype that comprises 3 X chromosome str locus site DXS7132, DXS6799 and DXS6804 is a template respectively, 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;
Wherein, the primer of DXS7132 gene locus pcr amplification is to being:
Upstream primer: agcccatttt cataataaat cc 22;
Downstream primer: fluorescence molecule-aatcagtgct ttctgtacta ttgg 24;
The primer of DXS6799 gene locus pcr amplification is to being:
Upstream primer: fluorescence molecule-atgaattcag aattatcctc atacc 25;
Downstream primer: gaaccaacct gcttttctga 20;
The primer of DXS6804 gene locus pcr amplification is to being:
Upstream primer: fluorescence molecule-cccagatatt ttgaccacca 20;
Downstream primer: ggcatgtggt tgctataacc 20;
2) the STR allelotrope of pcr amplification X chromosome to be measured:
With the genome that comprises X chromosome to be measured is template, with the primer of the str locus site pcr amplification of described 3 X chromosomes of step 1) to being primer, the STR allelotrope of 3 X chromosomes that comprise at same amplification system pcr amplification X chromosome to be measured;
3) fluoroscopic examination somatotype
With pcr amplification to the allelic ladder that has the fluorescence molecule mark and the STR allelotrope fragment of X chromosome to be measured carry out capillary electrophoresis separation; Adopt the fluorescence developing method, X chromosome to be measured is carried out the stdn somatotype according to the electrophoretic separation result.
2, the fluorescence detection method of X chromosome str locus as claimed in claim 1 site somatotype is characterized in that described fluorescence molecule is FAM, JOE, ROX, HEX or TMR.
3, the fluorescence detection method of X chromosome str locus as claimed in claim 1 site somatotype is characterized in that the parameter of described pcr amplification is: 94 ℃ of pre-sex change 6min, 94 ℃ of sex change 30 seconds, 60 ℃ of renaturation 35 seconds, 72 ℃ were extended 30 seconds, 30 circulations, last 72 ℃ are extended 10min.
4, the fluorescence detection method of X chromosome str locus as claimed in claim 1 site somatotype, it is characterized in that, the tumor-necrosis factor glycoproteins of described DXS7132 gene locus is cata, comprises 6 allelotrope, and the allelotrope clip size of pcr amplification is 278-298bp.
5, the fluorescence detection method of X chromosome str locus as claimed in claim 1 site somatotype, it is characterized in that, the tumor-necrosis factor glycoproteins of described DXS6799 gene locus is tatc, comprises 8 allelotrope, and the allelotrope clip size of pcr amplification is 230-258bp.
6, the fluorescence detection method of X chromosome str locus as claimed in claim 1 site somatotype, it is characterized in that, the tumor-necrosis factor glycoproteins of described DXS6804 gene locus is atag, comprises 10 allelotrope, and the allelotrope clip size of pcr amplification is 161-197bp.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886497A (en) * | 2014-12-15 | 2016-08-24 | 复旦大学 | Allelic ladder of polymorphic short tandem repeat (STR) loci as well as preparation method, identification method and application thereof |
| CN107273204A (en) * | 2016-04-08 | 2017-10-20 | 华为技术有限公司 | Resource allocation methods and device for genetic analysis |
| 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 |
| CN111893167A (en) * | 2020-08-10 | 2020-11-06 | 赛济检验认证中心有限责任公司 | Method for identifying sample ancestral source by STR gene detection method |
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2009
- 2009-07-21 CN CNA2009100233975A patent/CN101608233A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886497A (en) * | 2014-12-15 | 2016-08-24 | 复旦大学 | Allelic ladder of polymorphic short tandem repeat (STR) loci as well as preparation method, identification method and application thereof |
| CN107273204A (en) * | 2016-04-08 | 2017-10-20 | 华为技术有限公司 | Resource allocation methods and device for genetic analysis |
| CN107273204B (en) * | 2016-04-08 | 2020-10-09 | 华为技术有限公司 | Resource allocation method and device for gene analysis |
| US10853135B2 (en) | 2016-04-08 | 2020-12-01 | Huawei Technologies Co., Ltd. | Resource allocation method and apparatus for gene analysis |
| 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 |
| CN108676856B (en) * | 2018-04-17 | 2022-07-22 | 深圳华大法医科技有限公司 | Human X chromosome STR locus detection system, DNA sample analysis method and primer pair |
| CN111893167A (en) * | 2020-08-10 | 2020-11-06 | 赛济检验认证中心有限责任公司 | Method for identifying sample ancestral source by STR gene detection method |
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Open date: 20091223 |