CN1276427A - Process for preparing allelic gene step of shortly serial repetitive sequence - Google Patents

Abstract

A process for preparing allelic gene step reagent used in forensic medicine, archaeology, genetics and oncology includes such steps as extracting DNA fro human karyocyte, amplifying each STR site by PCR method to find the allelic gene type at each site, linking the purified PCR resultant to T carrier, extracting plasmid DNA, testing purity and quantity, and respectively preparing each gene fragments by STR-PCR technique. It features high preparing stability.

Description

The technology of preparing of STR allelic ladder

The invention belongs to genetically engineered, relate in particular to a kind of technology of preparing of STR allelic ladder, this technology can be applicable to individual recognition, paternity identification and the heredity in fields such as medical jurisprudence, archeology, genetics and oncology and the aspects such as diagnosis of neoplastic disease.

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.Wherein little satellite only is by what 2-7bp formed in repeating unit, thus be called again STR (Shorttandem repeat, SIR).The number of the genomic satellite DNA of different human body 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 human body is carried out individual recognition, paternity identification and heredity and neoplastic disease are diagnosed in recent years in fields such as medical jurisprudence, archeology, genetics and oncology, thereby the development in these fields of giving has brought new revolutionary variation.In this technology, position and discern in order to give each allelotrope exactly, must be provided with and the corresponding allelic ladder of each allelotrope (Allele Ladder) is marker (Markers).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 neighbouring lane under the identical deposition condition, compare after the dyeing, 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 several amplified productions of each allelotrope from each site screening of people's gene group, is directly used in AlleleLadder after the 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.

Zhi Bei allelic ladder has a lot of weak points as stated above:

When (1), directly selecting the people's gene group as amplification template, some band often occurring is repeat amplification protcol, and some band really is difficult to find corresponding amplification template, and the shade of each band of Zhi Bei allelic ladder differs thus, even zoning occurs lacking.

(2), to produce in batches, must often go to select and prepare and can represent the segmental template of all allelotrope, not only workload is 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.

(3), human gene group DNA's molecular weight is too big, structure is extremely complicated, is difficult to linearizing and unwinds, so the output of amplified production is lower, and non-specific amplification is more serious, to the purifying of product difficulty, is not suitable for batch process.

(4), if increase once more after the amplified production of personnel selection gene DNA mixes, its non-specific amplification is more serious, the color of each band heterogeneity more in the amplified production, this method more is not suitable for producing in batches.

(5), when the mixture of amplified production is directly used in allelic ladder, may occur degraded at short notice, make its each band smudgy, perhaps configuration changes to some extent, and its electrophoretic mobility is changed.Owing to the quality change that these reasons cause all can not be used as marker.

For above-mentioned reasons, still not having any unit so far can produce a kind of stable STR allelic ladder in batches and supply with relevant department and use.

Purpose of the present invention is exactly for a kind of technology of preparing of STR allelic ladder is provided, this technology can manufacture good stability and (was not promptly occurring the change of degraded and configuration below the normal temperature at least within 1 year, in the sex change of various concentration or non-sex change glue, all keep consistent) with the electrophoretic mobility of amplified production, both can be used for argentation, also can be used for the allelic ladder reagent of fluorescent method.

A kind of technology of preparing of STR allelic ladder is characterized in that comprising following production sequential steps:

1, from people's karyocyte, extracts DN/A: get anticoagulation 20 μ l, add erythrocyte cracked liquid D.5ml about, fully behind the mixing, centrifugal 5 minutes of 5000rpm, wash twice with erythrocyte cracked liquid again, precipitation adds 20 μ l write cell lysis buffers, behind the mixing, 100 ℃ were boiled 10 minutes, got 1 μ l and carried out the STR-PCR amplification;

2, increase by each STR site PCR method of publishing both at home and abroad, to seek the allelotype in each site;

3, QIAquickGel Extraction Kit (or like product of other company) specification sheets of producing by German QIAGEN company, fast purifying PCR product;

4, pGEM-TVector Systems (or like product of other company) specification sheets of producing by Promega company is connected to the PCR product of purifying on the T carrier;

5, extract plasmid DNA by the method for the 19th page of " molecular cloning " second edition Chinese edition, and carry out purity and quantitative test;

6, prepare each allelotrope fragment respectively with the STR-PCR technology, then amplified production is carried out purifying and quantitative assay;

7, the allelotrope fragment in each site is carried out sequential analysis, to measure each segmental bp number and series connection repeating unit number;

8,, add stablizer, and press the appropriate amount packing with after each fragment balanced mix;

9, carry out electrophoresis detection once more before dispatching from the factory.

The present invention has following characteristics:

1, each allelic template that is used to increase prepares by molecule clone technology, the sequence in the last same site of the dna sequence dna of this template and 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 having guaranteed these two kinds of amplified productions remains unchanged.

2, the length of the template that obtains by molecule clone technology is much 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 with 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.

3, each allelotrope fragment is to prepare one by one, measures its content then respectively, makes allelic ladder after the balanced mix again, is homogeneous with regard to the depth of having guaranteed every band color like this.

4, measure with the easier qualitative, quantitative of accomplishing of the template of molecule clone technology preparation, the template that one deutero-albumoseization is good can be used several years, this has not only simplified the loaded down with trivial details work of going to seek and extract each allelotrope template repeatedly from people's karyocyte widely, main is has avoided as much as possible because the difference of every batch of template quality, the negative impact of bringing for the stability of the stdn of producing and quality product.

5, when producing allelic ladder in batches, after the 5 ' end of one of the primer was put on fluorescein, just can obtain can be for the allelic ladder of fluoroscopic examination.

But 6, prepare allelic ladder not only mass, standardized production by this patent, and the stability of product is good especially, below the normal temperature at least within 1 year product do not have degraded and configuration change etc., its quality can reach the level of similar import reagent fully.

Describe in detail below.

The present invention includes following production sequential steps:

1, from people's karyocyte, extracts DNA: get anticoagulation 20 μ l, add about erythrocyte cracked liquid 0.5ml, fully behind the mixing, centrifugal 5 minutes of 5000rpm, wash twice with erythrocyte cracked liquid again, precipitation adds 20 μ l write cell lysis buffers, behind the mixing, 100 ℃ were boiled 10 minutes, got 1 μ l and carried out the STR-PCR amplification.

2, increase by each STR site PCR method of publishing both at home and abroad, to seek the allelotype in each site.With the VWA site is that example is described as follows: primer sequence is respectively 5 '-GGGATTTCCCTATGGATTGG-3 ' and 5 '-GCGAAAGAATGAGGACTACAT-3 '.If detect with fluorescent method, 5 ' of primer end is put on fluorescein therein.Each amplification sample comprises: 2-40ng human genome DNA, 1 * Taq damping fluid, 1.5mmol/L Mgcl, every kind of Nucleotide 150 μ mol/L, 1U Taq polysaccharase, every kind of primer 0.25 μ mol/L, reaction volume 25 μ l.95 ℃ of pre-sex change of elder generation are 2 minutes in thermal cycler, circulate then 30 times, and each 94 ℃ of sex change 1 minute were annealed 1 minute for 60 ℃, and 72 ℃ were extended 2 minutes.Carry out electrophoresis with 7.5% polyacrylamide, silver dyes then.The VWA genotype is determined in electrophoretic band position by comparative sample pcr amplification product and human VWA site Allele Ladde.

3, QIAquickGel Extraction Kit (or like product of other company) specification sheets of producing by German QIAGEN company, fast purifying PCR product.Concrete grammar is as follows: add in the PCR product of PE to 1 volume of 5 times of volumes, be added to after the mixing in the QIAquick post, this post is placed on the 2ml collection tube, quick centrifugal 30-60 second, discard centrifugate, be added to quick more centrifugal 30-60 second in the post with 0.75ml Buffer PE, discard centrifugate, post is placed on the new 1.5ml centrifuge tube, add an amount of water in post, centrifugal 1 minute fast, collection centrifugate was standby.

4, pGEM-TVector Systems (or like product of other company) specification sheets of producing by Promega company is connected to the PCR product of purifying on the T carrier, that is: 2 * T4 DNAligase beffer, 5 μ l, pGEM-T 1 μ l, T4 DNA liggase 1 μ l, purified PCR product is an amount of, adds water to 10 μ l4 ℃ connections then and spends the night, and is transformed in the intestinal bacteria again, from selecting picking list bacterium colony on the culture medium flat plate, therefrom select needed reorganization bacterium then.

5, extract plasmid DNA by the method for the 19th page of " molecular cloning " second edition Chinese edition, the reorganization bacterium that is about to the cultivation of LB liquid nutrient medium is transferred in the 1.5ml centrifuge tube, and centrifugal 5 minutes of 5000rpm abandons supernatant, add the solution I of 100 μ l precoolings, thermal agitation fully suspends it; And then add the solution II of the new preparation of 200 μ l; Cover the tight mouth of pipe, put upside down centrifuge tube fast and be placed on ice for 5 times; Add the ice-cold solution III of 150 μ l again, after 10 seconds of gentle vibration, placed again 3-5 minute on ice, centrifugal 10 minutes of 4 ℃ of 12000rpm; Supernatant is transferred in another centrifuge tube, added isopyknic phenol/chloroform, after the vibration evenly, centrifugal 2 minutes of 4 ℃ of 12000rpm; Supernatant is transferred in another centrifuge tube, added 2 times of volume of ethanol, fully behind the mixing, room temperature was transferred 2 minutes, centrifugal 5 minutes of 4 ℃ of 12000rpm; Wash plasmid DNA one time with 1ml 70% ethanol again, remove supernatant then, in air, make its drying; The TE that adds the Pancreatic RNase (20 μ g/ml) of an amount of no DNA enzyme dissolves plasmid DNA again.Use its purity of spectrophotometry and content then, and be kept at-20 ℃ standby.

6, prepare each allelotrope fragment respectively with the STR-PCR technology, when the allelic ladder of preparation band fluorophor, 5 ' of primer end is put on fluorescein earlier therein.Then amplified production is carried out purifying and quantitative assay.

7, the allelotrope fragment in each site is carried out sequential analysis, to measure each segmental bp number and series connection repeating unit number.

8,, add stablizer, and press the appropriate amount packing with after each fragment balanced mix.

9, carry out electrophoresis detection once more before dispatching from the factory.

Claims (9)

1. A kind of technology of preparing of STR allelic ladder is characterized in that comprising following production sequential steps:

   1, from people's karyocyte, extracts DNA: get anticoagulation 20 μ l, add about erythrocyte cracked liquid 0.5ml, fully behind the mixing, centrifugal 5 minutes of 5000rpm, wash twice with erythrocyte cracked liquid again, precipitation adds 20 μ l write cell lysis buffers, behind the mixing, 100 ℃ were boiled 10 minutes, got 1 μ l and carried out the STR-PCR amplification;
2. 2, increase by each STR site PCR method of publishing both at home and abroad, to seek the allelotype in each site;
3. 3, QIAquickGel Extraction Kit (or like product of other company) specification sheets of producing by German QIAGEN company, fast purifying PCR product;
4. 4, pGEM-TVector Syslems (or like product of other company) specification sheets of producing by Promega company is connected to the PCR product of purifying on the T carrier;
5. 5, extract plasmid DNA by the method for the 19th page of " molecular cloning " second edition Chinese edition, and carry out purity and quantitative test;
6. 6, prepare each allelotrope fragment respectively with the STR-PCR technology, then amplified production is carried out purifying and quantitative assay;
7. 7, the allelotrope fragment in each site is carried out sequential analysis, to measure each segmental bp number and series connection repeating unit number;
8. 8,, add stablizer, and press the appropriate amount packing with after each fragment balanced mix;
9. 9, carry out electrophoresis detection once more before dispatching from the factory.