CN1644709A - Individual DNA identification by short serial repeated sequential point isogenic gradient and determination reagent box - Google Patents
Individual DNA identification by short serial repeated sequential point isogenic gradient and determination reagent box Download PDFInfo
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Abstract
An identification method and its regent box for recognizing the unit DNA by the allelic ladder of short series-wound repeating serial locus have been opened the invention. The process is: Screening the SRT locus, designing the common reading things for synthesizing special PCR, then to mark the leading things, respectively. Then to extract the DNA of the human, going on the PCR reaction, detecting the part by electrophoresis and purifying the part. Last to connect the product of PCR to the carrier, then getting the clone of the different genes, making the every allele PCR reaction by using the leading thing of fluorescent number, mixing the PCR products of allele for STR locus then adding to the stabilizer. So we can get the allelic ladder. The ladder is the regent box to recognize the DNA. The invention needn't to collect the different gene blood again and again. And the box cost little. It is easy to make, also have good stability.
Description
One, technical field:
The present invention relates to gene engineering technology field, can be used for individual recognition, paternity identification and the heredity in fields such as forensic biology, anthropology, genetics and oncology and the diagnosis aspect of tumour, a kind of fluoroscopic examination MULTIPLE COMPOSITE str locus site that is applicable to that belongs to the preparation of using gene engineering method especially, to its STR allelic ladder that carries out gene type, be used for the individual recognition DNA detection and identify.
Two, background technology:
The human genome DNA has 3 * 10
9Bp, wherein 10% is tandem repetitive sequence, is called as satellite DNA.Length by repeating unit can be divided into large satellite, middle satellite, moonlet and little satellite again.Little satellite of only forming of repeating unit wherein by 2～7bp, be called again STR (short tandem repeat, STR).The number of the genomic satellite DNA of Different Individual repeating unit is variable, has therefore formed extremely complicated allelotrope fragment length polymorphism.According to this rule, set up a kind of brand-new STR-PCR (STR polymerase chain reaction) technology that individuality is carried out individual recognition, paternity identification and heredity and tumour are diagnosed in medical jurisprudence method, archeology, genetics and oncology in recent years.In this technology, position and discern in order to give each allelotrope accurately, (Allelic ladder AL) is marker (Markers) with the corresponding allelic ladder of each allelotrope in necessary setting.Because of the length of each gene fragment in the allelic ladder all is known, it is carried out electrophoretic separation with the amplified production of each testing sample on the swimming lane under the identical deposition condition, compare, just be easy to judge the genotype of testing sample, so allelic ladder is most important in the result of STR-PCR judges.
The method of each STR site allelic ladder of present domestic preparation has two kinds: a kind of is can represent the segmental different genotype sample amplification of each allelotrope from each site screening of people's gene group, and the different fragments product that obtains is directly used in allelic ladder after mixing; Another kind method is to represent all allelic amplified production mixtures on certain site, and amplification once more after the dilution is used as allelic ladder with the product that increases once more.The weak point of these two kinds of methods:
1, some STR genetic marker site genetic diversity in Chinese population (Mongolian race) is lower, and the polymorphism information amount is less, is not suitable for East Asia and Southeast Asian when the individual recognition DNA detection.
When 2, directly selecting the human gene group DNA as amplification template, because each allelic length difference, be prone to the advantage pcr phenomenon, be that the short allelotrope of length is amplified easily, the allelotrope that length is long then is difficult for being amplified, and makes the amount difference of amplified production, the allelic amount of each of Zhi Bei allelic ladder differs thus, even occur not increasing, promptly there is not amplified production, can't detected phenomenon.
3, will produce in batches, must often go to select and prepare and can represent the segmental template of all allelotrope, not only workload be big, and the purity of the same template of each usefulness and content etc. all have discrepancy, makes its amplification condition be difficult to stdn.
4, human gene group DNA's molecular weight is big, structure and complexity thereof, and as easy as rolling off a log non-specific amplification to occur more serious, is not suitable for producing in batches.
5, human gene group DNA's amplified production increases after mixing once more, because the PCR product reduces with respect to the dna fragmentation biological activity in microbial culture and the tissue, its non-specific amplification is more serious.This method is not suitable for producing in batches.
When 6, the mixture of amplified production is directly used in allelotrope, may occur degraded at short notice, make its each band smudgy, perhaps configuration changes to some extent its electrophoretic migration is changed.Owing to the quality change that these reasons cause all can not be used as marker.
7, the detection method of the allelic ladder of domestic production at present use mainly is an argentation, this method trivial operations, and the background height, detection sensitivity and resolving power are all lower, easily produce result uncertain even erroneous judgement.The operating technology means of high request, seriousness of identifying etc. for legal medical expert are fallen behind relatively.
8, Zhi Bei allelic ladder is one to three each allelic mixing of STR site, does not detect when having more a plurality of STR of realization site.
Three, summary of the invention:
In order to overcome the defective that prior art exists, the invention provides a kind of individual recognition DNA authentication method and detection kit thereof with STR site allelic ladder.It need not to collect repeatedly the blood specimen with different genotype individuality, can reduce cost effectively.
The present invention realizes that by following technical scheme the inventive method adopts the step of the technology of preparing of three fluorescence mark STR site allelic ladder:
Allele distributions and the frequency thereof of investigation STR site in Chinese population and other people group;
Screen 16 to 20 STR sites and be used for the individual recognition DNA detection;
The specific PCR amplification general primer in synthetic selected each the STR site of design and grouping fluorescent dye primer, fluorescent mark divides three groups, is respectively blue, green, yellow;
Collect the not homoallelic human tissue sample that contains selected each STR site;
From people's karyocyte, extract the human gene group DNA;
Utilize the DNA and the synthetic general primer that extract respectively the PCR reaction to be carried out in each STR site;
The electrophoresis detection amplified fragments, the rubber tapping purifying;
The PCR product is connected on the carrier, obtains the not homoallelic clone in each STR site;
Mono-clonal is transformed in the intestinal bacteria, obtains containing each allelic plasmid;
Utilize plasmid template that each allelotrope is carried out PCR respectively with corresponding fluorescent dye primer, prepare each different allelotrope;
Fluorescent value according to each pcr amplification product detects quantitatively each allelotrope;
Equimolar amount mixes each allelic pcr amplification product in each STR site in proportion, measures with fluorescence detection, and the ratio of regulating each product adds the stable reagent agent, can obtain allelic ladder.
Above-mentioned described electrophoresis system adopts genetic analyzer (sequenator) capillary electrophoresis or polyacrylamide gel electrophoresis, and LASER Excited Fluorescence signal, CCD detect collects signal.
Above-mentioned STR site can relate to all the STR sites on human 46 karyomit(e)s, promptly comprises the STR site on euchromosome, X chromosome and the Y chromosome.
Above-mentioned primer is the primer of fluorochrome label and the primer of non-fluorochrome label, and the length of these primers is 15～40 Nucleotide.
A kind of detection kit that forms with the individual recognition DNA authentication method of STR site allelic ladder is provided with in this box:
Many group fluorescent mark allelic ladders;
The primer and the unlabelled primer of the three fluorescence dye marker that one cover is corresponding with each STR site;
Red fluorescence marker DNA molecular weight inner reference standard;
PCR reacts each component;
Gold medal Taq enzyme;
Positive control dna.
The present invention has following characteristics:
1, the present invention is chosen in 16 to 20 STR sites that the height polymorphism is arranged among Chinese population and other crowd, and very high specific aim, specificity are arranged, associating resolving power and individual recognition ability height.
2, the allelic ladder of the present invention's preparation is the multicolor fluorescence mark.
3, the self-built multiplex PCR composite amplification system of the present invention has stability, high efficiency, repeatability.
Check and analysis when 4, multicolor fluorescence mark and multiplex PCR composite amplification system have been realized a plurality of STR site.
5,, own unique design of primers and fluorescent mark manufacturing process are arranged for selected 16 to 20 sites.
6, each allelic template that is used to increase prepares by molecule clone technology, the dna sequence dna of this template is through sequence verification, the sequence that goes up same site with the human gene group DNA is identical, therefore the dna sequence dna and the configuration of the amplified production that obtains with these two kinds of different templates are in full accord, thereby the electrophoretic mobility of these two kinds of amplified productions is consistent, and science is accurate more to have guaranteed the gene type result.
7, the template length that obtains by molecule clone technology is far smaller than human gene group DNA's length, easily linearizing and unwinding, under the same conditions, the amplified production that obtains is far above the amount of human gene group DNA's amplification, and the non-specific amplification product is also far fewer than human gene group DNA's amplified production, so easily mass-produced.
8, each allelotrope fragment prepares one by one, measures its content then respectively, and equimolar amount is made allelic ladder after mixing in proportion again, has so just guaranteed the consistence of each allelotrope peak height.
9, measure with the easier qualitative, quantitative of accomplishing of the template of molecule clone technology preparation, once standardized template can be used for many years, this has not only simplified the loaded down with trivial details work of going to seek each allelotrope template of nuclear extraction repeatedly from people's karyocyte widely, main making avoided as much as possible because the difference of every batch of template quality, make production be more prone to stdn and quality product is more stable.
10, the present invention has adopted unique fluorescence molecule amount internal standard preparation, uses PCR method, has improved the stability and the output of product.
The present invention also has following advantage except the advantage with prior art:
1, need not to collect repeatedly blood specimen with different genotype individuality;
2, make simple flow, the cycle is short, and is simple to operate;
3, cost of manufacture is low, and is economical and practical;
4, test kit can manufacture, good stability.
Four, embodiment:
A kind of individual recognition DNA authentication method with STR site allelic ladder, the concrete steps of the technology of preparing of employing three fluorescence mark STR site allelic ladder:
1, from people's karyocyte, extracts DNA: get anticoagulation 20ul, add about erythrocyte cracked liquid 0.5ul, fully behind the mixing, centrifugal 5 minutes of 5000rpm washes twice with erythrocyte cracked liquid again, and precipitation adds the 20ul write cell lysis buffer, behind the mixing, 100 ℃ were boiled 10 minutes, got 1ul and carried out pcr amplification.
2, the specific PCR amplification general primer in synthetic selected each the STR site of design and grouping fluorescent dye primer, fluorescent mark divides three groups, is respectively blue, green, yellow.
3, increase according to each STR site PCR method of publishing both at home and abroad, to seek the allelotype in each site.
4, the PCR product is through agarose gel electrophoresis, EB dyeing.
5, reclaim dna fragmentation with the dialysis tubing electroelution method, with phenol-chloroform extracting and purifying DNA fragment.
6, with TAKARA T-Vector test kit DNA is connected on the carrier, the carrier that will contain dna fragmentation is transformed in the intestinal bacteria, incubated overnight, i.e. PMD18-T Vector 0.5ul, Solution 1 2.5ul, purified PCR product is an amount of, add water to 20ul, 4 ℃ are spent the night, and are transformed in the intestinal bacteria again, from selecting picking list bacterium colony on the culture medium flat plate, therefrom select needed reorganization bacterium then.
7, extract plasmid according to Shen friend's test kit (or like product of other companies) specification sheets: the reorganization bacterium that is about to the cultivation of LB liquid nutrient medium is transferred in the 2ml centrifuge tube, and centrifugal 30 seconds of top speed is abandoned supernatant, adds 250ulS1 liquid, and vibration is to thoroughly suspending; Add 250ulS2 solution, leniently spin upside down centrifuge tube 5～10 times immediately with mixing; The S3 liquid that adds the 400ul4oC precooling, gentle immediately and spin upside down centrifuge tube 5～10 times fully with mixing, room temperature leaves standstill 2min, high speed centrifugation 10min; Supernatant is transferred in the DNA adsorption column, and centrifugal 2min abandons worry liquid; Add 500ulW1 solution, centrifugal 30s abandons worry liquid; Add 700ulW2 liquid, centrifugal 30s abandons worry liquid; Add 700ulW2 liquid again, centrifugal 30s abandons worry liquid; Adsorption column is put into same collection tube, high speed centrifugation 1min; Move in the new 1.5ml centrifuge tube, central authorities add the 60ul elutriant at the DNA adsorption film, and room temperature leaves standstill 1min, and centrifugal 1min eluted dna is preserved standby.
8, utilize plasmid template that each allelotrope is carried out PCR respectively with corresponding fluorescent dye primer, prepare each different allelotrope.
9, the fluorescent value according to each pcr amplification product detects quantitatively each allelotrope.
10, molar weight is mixed each allelic pcr amplification product in each STR site, measures with fluorescence detection, and the ratio of regulating each product adds the stable reagent agent, can obtain allelic ladder.
The three fluorescence mark STR site allelic ladder that has by method for preparing is used for individual recognition DNA fluorescence detection reagent kit, and this test kit is equipped with following component:
Many group fluorescent mark allelic ladders;
The primer and the unlabelled primer of the three fluorescence dye marker that one cover is corresponding with each STR site;
Red fluorescence marker DNA molecular weight inner reference standard;
PCR reacts each component;
Gold medal Taq enzyme;
Positive control dna.
More than each component independence packing, preceding two kinds of the present invention illustrate that other four kinds of components are marketing, during use routinely PCR method carry out, electrophoresis detection on genetic analyzer is collected signal, analytical results.
Claims (5)
1. individual recognition DNA authentication method with STR site allelic ladder is characterized in that adopting the step of the technology of preparing of three fluorescence mark STR site allelic ladder:
Allele distributions and the frequency thereof of investigation STR site in Chinese population and other people group;
Screen 16 to 20 STR sites and be used for the individual recognition DNA detection;
The specific PCR amplification general primer in synthetic selected each the STR site of design and grouping fluorescent dye primer, fluorescent mark divides three groups, is respectively blue, green, yellow;
Collect the not homoallelic human tissue sample that contains selected each STR site;
From people's karyocyte, extract the human gene group DNA;
Utilize the DNA and the synthetic general primer that extract respectively the PCR reaction to be carried out in each STR site;
The electrophoresis detection amplified fragments, the rubber tapping purifying;
The PCR product is connected on the carrier, obtains the not homoallelic clone in each STR site;
Mono-clonal is transformed in the intestinal bacteria, obtains containing each allelic plasmid;
Utilize plasmid template that each allelotrope is carried out PCR respectively with corresponding fluorescent dye primer, prepare each different allelotrope;
Fluorescent value according to each pcr amplification product detects quantitatively each allelotrope;
By each the allelic pcr amplification product that mixes each STR site than row equimolar amount, to measure with fluorescence detection, the ratio of regulating each product adds the stable reagent agent, can obtain allelic ladder.
2. the individual recognition DNA authentication method with STR site allelic ladder according to claim 1, it is characterized in that: described electrophoresis system adopts genetic analyzer (sequenator) capillary electrophoresis or polyacrylamide gel electrophoresis, LASER Excited Fluorescence signal, CCD detect collects signal.
3. the individual recognition DNA authentication method with STR site allelic ladder according to claim 1, it is characterized in that: described STR site can relate to all the STR sites on human 46 karyomit(e)s, promptly comprises the STR site on euchromosome, X chromosome and the Y chromosome.
4. the individual recognition DNA authentication method with STR site allelic ladder according to claim 1, it is characterized in that: primer is the primer of fluorochrome label and the primer of non-fluorochrome label, and the length of these primers is 15～40 Nucleotide.
5. detection kit that is formed by claim 1 individual recognition DNA authentication method is characterized in that: be provided with following component in the individual recognition DNA fluorescence detection reagent kit of this three fluorescence mark STR site allelic ladder:
Many group fluorescent mark allelic ladders;
The primer and the unlabelled primer of the three fluorescence dye marker that one cover is corresponding with each STR site;
Red fluorescence marker DNA molecular weight inner reference standard;
PCR reacts each component;
Gold medal Tag enzyme;
Positive control dna.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410025157 CN1644709A (en) | 2004-06-15 | 2004-06-15 | Individual DNA identification by short serial repeated sequential point isogenic gradient and determination reagent box |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410025157 CN1644709A (en) | 2004-06-15 | 2004-06-15 | Individual DNA identification by short serial repeated sequential point isogenic gradient and determination reagent box |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559878A (en) * | 2011-12-21 | 2012-07-11 | 北京阅微基因技术有限公司 | Composite amplification system and detection kit of mouse short tandem repeat |
| CN103065067A (en) * | 2012-12-26 | 2013-04-24 | 深圳先进技术研究院 | Method and system for filtering sequence segments in short-sequence assembly |
| CN106566827A (en) * | 2016-11-07 | 2017-04-19 | 江苏苏博生物医学股份有限公司 | Novel dye labeling method |
| CN108085367A (en) * | 2018-01-26 | 2018-05-29 | 公安部第研究所 | A kind of genetic analyzer tests special allele standard control preparation method of reagent thereof |
| 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 |
-
2004
- 2004-06-15 CN CN 200410025157 patent/CN1644709A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559878A (en) * | 2011-12-21 | 2012-07-11 | 北京阅微基因技术有限公司 | Composite amplification system and detection kit of mouse short tandem repeat |
| CN102559878B (en) * | 2011-12-21 | 2013-09-25 | 北京阅微基因技术有限公司 | Composite amplification system and detection kit of mouse short tandem repeat |
| CN103065067A (en) * | 2012-12-26 | 2013-04-24 | 深圳先进技术研究院 | Method and system for filtering sequence segments in short-sequence assembly |
| CN103065067B (en) * | 2012-12-26 | 2016-07-06 | 深圳先进技术研究院 | The filter method of sequence fragment and system in short sequence assembling |
| CN106566827A (en) * | 2016-11-07 | 2017-04-19 | 江苏苏博生物医学股份有限公司 | Novel dye labeling method |
| CN106566827B (en) * | 2016-11-07 | 2019-12-17 | 江苏苏博生物医学股份有限公司 | dye marking method |
| CN108085367A (en) * | 2018-01-26 | 2018-05-29 | 公安部第研究所 | A kind of genetic analyzer tests special allele standard control preparation method of reagent thereof |
| 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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