CN102251032B - Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat) - Google Patents
Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat) Download PDFInfo
- Publication number
- CN102251032B CN102251032B CN2011101858340A CN201110185834A CN102251032B CN 102251032 B CN102251032 B CN 102251032B CN 2011101858340 A CN2011101858340 A CN 2011101858340A CN 201110185834 A CN201110185834 A CN 201110185834A CN 102251032 B CN102251032 B CN 102251032B
- Authority
- CN
- China
- Prior art keywords
- str
- dna
- reference material
- plasmid
- recombinant plasmid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of a reference material (RM) for detecting a human DNA (deoxyribonucleic acid) STR (short tandem repeat), and the method is used for preparing the RM for detecting the human DNA STR by utilizing a molecular cloning technique. The preparation method is mainly characterized by comprising the following steps: recombining artificially synthesized or PCR amplified fragments containing STR gene loca with plasmids, transferring the recombinant plasmids into cells, screening out clones containing correct insert fragments by sequencing, and performing mass propagation; and extracting the recombinant plasmids, performing enzyme digestion, purifying and mixing to obtain a large quantity of high-quality RMs for detecting the DNA STR. The preparation method has the advantages of simple preparation process and low cost; and the RM for detecting the DNA STR prepared by the method is good in genetic stability and easy for storage.
Description
Technical field
The present invention relates to biological technical field, be particularly related to a kind of human DNA STR and detect preparation method, be used to solve the accuracy and the standardization issue of DNA detection of individual recognition, paternity test and the gene diagnosis aspect in fields such as medical jurisprudence, anthropology, genetics and disease with reference material.
Background technology
Reference material is the measurement standard with accurate value, according to " international metrology basic terms " and " International Standards Organization's guide 30 ", reference material (reference material, RM) just like giving a definition: have one or more enough all even characteristic values of having determined well, in order to the calibration measurement device, estimate measuring method or give a kind of material or the material of material assignment.Realized the transmission of value and traced to the source by reference material, made the standard of measuring to trace to the source to international fundamental unit.
DNA reference material (DNA standard reference materials, be called for short SRM), it is DNA measurement standard with characteristic value, can guarantee the validity and the comparability of DNA measuring result, the assurance measuring result can be traced to the source to International System of Units or other international consistent units of approving, guarantees the comparability and the consistence of the NA of D. Lab detected result.
DNA detection belongs to the category of biotechnology, and fast-developing biotechnology detects validity and has higher requirement, and biometric has also obtained people's attention as a brand-new science.In October, 1999, biometrics is brought in the 21st Conference General des Poids et Measures decision into schedule.Subsequently, (the Consultative Committee for Quantity ofSubstance-Metrology of amount of substance consultative committee, CCQM) (BioanalysisWorking Group BAWG), held 11 meetings to 2007 to have set up bioanalysis working group.The main purpose of this working group is to set up the biotechnology measuring system of can tracing to the source, top-priority is the stdn of nucleic acid measurement, protein measurement and method thereof, analytic process, and the development of reference material, so that consolidate the Application Areas of measuring with nucleic acid and protein.
DNA detection mainly adopts PCR method to detect, human DNA detects the human DNA genome that normally has the characteristic value with reference material, carry out PCR with reference material as template with the human DNA detection, the assurance of tracing to the source of science can be provided for DNA detection work, to improve the science and the reliability of detected result, also can test and calibrate, estimate and study the internal soundness control of the new DNA method of inspection, Laboratory Accreditation, DNA analysis, can also be used for examination DNA reviewer level and ability to instrument.
At the forensic dna detection range, its detection technique develops rapidly, and the stdn of testing process and standardization seem and be even more important that this just needs reference material that testing process is carried out quality control.1998, the DNA suggestion council standard 9.5 of FBI (FBI) is pointed out: " reference material (SRM) that laboratory every year or experiment condition all should use American National Standard technical institute (NIST) to provide when changing was verified the DNA checking procedure." I enclose and also am not fit to the reference material that forensic dna detects at present.
Forensic dna detects and has experienced restriction fragment length polymorphism (RFLP), STR (STR) and single nucleotide polymorphism (SNP).Equally, the forensic dna reference material is also constantly released new reference material along with the development of detection technique.NIST is responsible for exploitation country and international reference materials as the commercial department of the U.S..In the reference material that NIST provides, the somatotype that has the multiple standards material to be used for forensic dna detects---based on the SRM2390 of RFLP typing method, based on the SRM2391b of STR typing method, be used for SRM2392 that Mitochondrial DNA detects, be used for the SRM2395 of Y chromosome dna typing and be used for the quantitative SRM2372 of human DNA.
The genetic marker that is most commonly used to forensic dna check at present is the STR genetic marker, and (short tandem repeat is extensively to be present in the dna sequence dna that a class in the human genome has length polymorphism STR) to STR.It as core sequence, is the series connection repeated arrangement by 2-6 base, and its length polymorphism is mainly different with multiplicity and produce by the number of variations of core sequence.STR distributes extensively, number is many, account for 10% of human genome, it is generally acknowledged, the average every 6-10kb of human genome just has 1 str locus seat, the combination results of different str locus seats very abundant locus figure spectrum information, for the individual recognition in the forensic medicine in appraisal of material evidence and paternity test provide the foundation.Forensic dna STR detects with reference material and is used for quality control and quality-guarantee to the DNA detection process, by using reference material, make the stdn and the standardization of testing process, set up standardized DNA analysis technology, guarantee forensic dna check and analysis result's accuracy, fairness and reliability, for the detected result of DNA provides quality assurance, the DNA reference material also helps the interchange of the data between each laboratory, various countries.
American National Standard technical institute (NIST) has issued STR nineteen ninety-five and has detected with reference material-SRM2391, SRM2391 only contains the message of 4 str locus seats (TH01, F13A01, vWA and FES/FPS), this can not satisfy the STR typing method far away and develop rapidly the demand of STR detection with reference material, the STR message of all DNA samples that provided should be provided SRM2391, should comprise 13 core gene seats among the CODIS of FBI at least.In June, 1998, NIST carried out analyzing and testing again to SRM2391,25 locus somatotype information of each component have been detected, comprise 13 core gene seats and other 8 str locus seats (CSP1PO, D2S 1338, D3S 1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, F13A01, F13B, FES/FPS, FGA, LPL, Penta D, Penta E, TH01, TPOX and vWA) among the CODIS, and HLA-DQAI, PolyMarker, D1S80 and Amelogenin locus.After the analyzing and testing, NIST has issued the str locus somatotype definite value of SRM2391, the new DNA reference material SRM2391a that detects at STR that releases, it comprises cellular component and the genomic dna component of SRM2391, and removed amplified production, D1S80 allelic gene typing mixture and molecular weight marker, contain from 9947A and 9948 clones are extracted and quantitatively be the genomic dna of 1ng/ μ L.The DNA reference material SRM2391b that in January, 2003, NIST released again at the STR detection substitutes SRM2391a in the past, and SRM2391b contains all the str locus seats among the SRM2391a, has increased the SE33 locus in addition.
Reference materials such as the human genome 9947A, 9948, the K562 that provide in business-like amplification kit have played the effect of internal soundness control as the positive control of pcr amplification to DNA detection.But, the reference material that use in most of DNA detection laboratory is all provided by American National Standard technical institute (NIST), the DNA that its reference material contains proposes from the human cell, adopts the human cell to prepare the DNA reference material and has following shortcoming:
1, the heredity of cell is unstable.Cell cultures certain algebraically that goes down to posterity, chromosome number and structure can change, and hypodiploid, hyperdiploid or polyploid occur, and base deletion takes place or inserts sudden change in the DNA site.
2, the cell reserve capacity is big.Because cytogenetic unstable, needing the cultivation of cell to go down to posterity needs to keep enough backups in the process, causes the cell reserve capacity big.
3, cell culture condition requires high.Each factor such as temperature, gas phase, pH value, osmotic pressure etc. in the environment that the cell of will controlling well during culturing cell generates, in addition, also need with the direct contactee of cell such as culturing bottle, substrate, nutrient chemical etc. or indirect contact person as no cytotoxicities such as bottle cap bottle stoppers, if the tool cytotoxicity can cause the death of cell, avoid microbiological contamination, the crossed contamination of different cell types and the pollutions of objectionable impurities such as bacterium equally.
Therefore, the present invention adopts molecule clone technology to prepare human DNA STR detection reference material.
Summary of the invention
Detect the demanding problem of cytogenetics instability, culture condition that exists with the standard reference materials preparation process at the DNA STR of present use, an object of the present invention is to provide a kind ofly be easy to preserve, DNA STR that heritability is stable detects the preparation method with reference material.
The human DNA STR for preparing provided by the invention detects the method for using reference material, may further comprise the steps:
Recombinant plasmid in the positive colony that step 3, extraction step 2 are identified, the recombinant plasmid equal proportion that will contain different genotype is mixed, and the DNA STR that is configured to human genome detects and uses reference material.
As preferably, described target gene fragment is 5～200 kinds, and every kind of target gene fragment contains at least one genotype.
More preferably, described target gene fragment is 10～50 kinds, and every kind of target gene fragment contains at least one genotype.
As preferably, the described cell of step 2 is a Bacillus coli cells, and described plasmid vector is the corresponding plasmids of intestinal bacteria.
As preferably, the described recombinant plasmid of step 3 is a linear plasmid.
As preferably, the described target gene fragment of step 1 is for containing the fragment of str locus seat CSFlPO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta D, Penta E, Th01, Tpox, vWA and sex marker site Amelogenin respectively.Carry out pcr amplification with primer sequence shown in the SEQ ID No.1-SEQ ID No.42.
The present invention also provides the human DNA STR of described method preparation to detect and uses reference material.
Another object of the present invention provides the test kit of the human DNA STR reference material that comprises foregoing description.
The inventor selects 19 str locus seats (CSFlPO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta D, Penta E, Th01, Tpox, vWA) and sex marker site Amelogenin to prepare STR detection reference material.Can satisfy in the forensic science requirement substantially to the detection of above-mentioned 19 str locus seats and sex marker site Amelogenin for DNA detection in individual's identification and the paternity test.
Above-mentioned 19 str locus seats and sex marker site Amelogenin complete sequence that the inventor finds from GenBank and pertinent literature, it is several to primer to be with the sequence that finds that template is passed through Primer Premier software design, filters out locus of a pair of primer amplification with Oligo software then.When design STR detects primer with reference material, must guarantee that tumor-necrosis factor glycoproteins in the locus is positioned at the mid-way of amplified fragments, and apart from forward and reverse primer all between 400-600bp.Because two the site homologys of X, Y among the sex marker site Amelogenin are not very high, therefore need respectively design a pair of primer at two sites of X, Y.The primer of design adds restriction enzyme site at 5 ' end, helps the PCR product is inserted in the plasmid pUC18.The primer sequence of design sees Table 1.
The primer sequence of table 1. design
Annotate: underscore is the restriction enzyme site sequence.
Utilize above-mentioned primer to carry out polymerase chain reaction (PCR) amplification and obtain goal gene, according to the different restriction enzyme site of primer of design, plasmid pUC18 and purified PCR product are carried out double digestion with corresponding enzyme again.With the connection of spending the night of T4 ligase enzyme, change e. coli jm109 with the pUC18 behind the identical enzyme double digestion and PCR product over to, the picking transformant is with bacterium colony PCR and extract two kinds of methods of plasmid and identify and check order.
19 str locus seats searching that the present invention need clone in genome K562,9947A and 9948 and the genotype of sex marker site Amelogenin by analyzing sequencing result, find and contain all genotypic recon sequencing results.The bacterium liquid that will contain these recons is preserved.In addition, choose the bacterium liquid of these preservations, incubated overnight is extracted recombinant plasmid.By analyzing the sequence of each recon, selection is away from the carrier multiple clone site and have only the restriction enzyme single endonuclease digestion recombinant plasmid of a restriction enzyme site (Sca I or Aat II) in recombinant plasmid, promptly contain locus D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, FGA, Penta E, Th01, Tpox, the recombinant plasmid of vWA and sex marker site Amelogenin is cut with restriction enzyme Sca I enzyme, contain locus Penta D, D21S11, the recombinant plasmid of CSFlPO and D2S1338 is cut with restriction enzyme A at II enzyme, make recombinant plasmid be wire, enzyme is cut the back purifying.
Recombinant plasmid carries out the homogeneity (PCR output is identical) of single endonuclease digestion PCR product when helping others and being template PCR with recombinant plasmid, if do not carry out single endonuclease digestion, plasmid is superhelix, ring-type and three kinds of forms of wire, and these three kinds of form ratios be can not determine, the Taq enzyme is difficult for combining with superhelix, cyclic plasmid in the PCR process, causing with three kinds of form plasmids is that template PCR efficient is inconsistent, thereby influences the homogeneity of PCR product.Behind the single endonuclease digestion plasmid, plasmid all is wire, PCR efficient unanimity.
The recombinant plasmid that above-mentioned purifying is good with the Restriction Enzyme single endonuclease digestion, the genotype that comprises according to 20 locus among the 1ng/ μ L human genome K562,9947,9948 and contain this genotypic molar weight, the different genotypic plasmid equal proportions that contains is mixed, be configured to 3 cover forensic dna reference materials.
The present invention adopts molecule clone technology to prepare human DNA STR detection reference material, its maximum characteristics are to design primer voluntarily, synthetic or pcr amplification are obtained fragment and the plasmid reorganization that contains the str locus seat, recombinant plasmid is changed in the e. coli jm109, filter out by order-checking and to contain the segmental genotype of correct insertion, and preserve bacterium liquid.Can breed in a large number by bacterium, extract recombinant plasmid, by single endonuclease digestion, purifying, be mixed with out a large amount of high-quality STR somatotype reference materials.
The STR of the present invention's preparation detects with reference material and compares with reference material with cultivate preparation STR detection by the human cell, and major advantage is as follows:
1) the present invention utilizes the method for molecular cloning, the human STR somatotype reference material of preparation in cell, and preparation process is simple, cost is low, require low to personnel's level;
2) STR of preparation method's acquisition of the present invention detects with reference material and compares with reference material with cultivate preparation STR detection by the human cell, has good genetic stability;
3) prepared bacterium liquid and the STR detection of the present invention is easy to preserve with reference material.
Description of drawings
Fig. 1 is preparation method's overall procedure of the present invention;
Fig. 2 is the pcr amplification product electrophorogram;
M：DNA?100bp?ladder；1：K562-D2S1338；2：9947A-D2S1338；3：9948-D2S1338；
4：K562-D3S1358；5：9947A-D3S1358；6：9948-D3S1358；
7：K562-D8S1179；8：9947A-D8S1179；9：9948-D8S1179；10：
K562-D5S818；11：9947A-D5S818；12：9948-D5S818；13：
K562-D21S11；14：9947A-D21S11；15：9948-D21S11；16：
K562-D18S51；17：9947A-D18S51；18：9948-D18S51；
19：K562-TPOX；20：9947A-TPOX
Fig. 3 is the pcr amplification product electrophorogram;
M：DNA?100bp?ladder；1：K562-D16S539；2：9947-D16S539；
3：9948-D16S539；
4：K562-THO1；5：9947-THO1；6：9948-THO1；
Fig. 4 is the pcr amplification product electrophorogram;
M：DNA?100bp?ladder；1：K562-Csf1Po；2：9947-Csf1Po；
3：9948-Csf1Po；4：K562-vWA；5：9967-vWA；6：9948-vWA；
7：K562-Amelogenin-Y；8：9947-Amelogenin-Y；
9：9948-Amelogenin-Y；10：K562-D6S1043；11：9947-D6S1043；
12：9948-D6S1043；13：K562-Amelogenin-X；14：9947A-
Amelogenin-X；15：9948-Amelogenin-X
Fig. 5 is the pcr amplification product electrophorogram;
M：DNA?100bp?ladder；1：K562-D13S317；2：9947-D13S317；
3：9948-D13S317；
4：K562-Penta?E?5：9947-Penta?E；6：9948-Penta?E；7：K562-Fga；
8：9947-Fga；
9：9948-Fga；10：9948-Tpox；
Fig. 6 is the pcr amplification product electrophorogram;
M：DNA?100bp?ladder；1：K562-D7S820；2：9947-D7S820；
3：9948-D7S820；
4：K562-D19S433；5：9947-D19S433；6：9948-D19S433；
7：K562-D12S391；8：9947-D12S391；9：9948-D12S391
Fig. 7 is the pcr amplification product electrophorogram;
M：DNA?100bp?ladder；1：K562-vWA；2：9947-vWA；3：9948-vWA；
4：K562-Penta?D；5：9947-Penta?D；6：9948-Penta?D
Fig. 8 is plasmid double digestion rear electrophoresis figure;
1:pUC18; 2:pUC18 (Pst I/EcoR I double digestion); 3:pUC18 (Hind III/PstI double digestion); 4:pUC18 (XbaI/EcoRI double digestion); 5:pUC18 (XbaI/PstI double digestion); 6:pUC18 (Pst1/BamHI double digestion); 7:pUC18 (XbaI/HindIII double digestion); 8:pUC18 (EcoR I/Hind III double digestion); 9:pUC18 (XbaI/BamHI double digestion)
Fig. 9 is a bacterium colony PCR electrophorogram;
1,10: be respectively regular-PCR product 9947-THO1,9947-D21S11; 2-9,11-22: be respectively bacterium colony PCR product 9947-THO1-(1-4), 9948-THO1-(1-4),
K562-D21S11-(1-4)、9947-D21S11-(1-4)、9948-D21S11-(1-4)；
Figure 10 is a bacterium colony PCR electrophorogram
1,6,15: be respectively regular-PCR product K562-D2S1338, K562-D3S1358,9947-D8S1179; 2-5,7-14,16-23: be bacterium colony PCR product 9948-D2S1338-(1-4), K562-D3S1358-(1-4), 9947-D3S1358-(1-4), K562-D8S1179-(1-4), 9948-D8S1179-(1-4)
Figure 11 is the plasmid electrophorogram;
1:PUC18 (contrast); 2-5:K562-D18S51-(1-4); 6-9:
9947A-D18S51-(1-4)；10-13：9948-D18S51-(1-4)；14-17：
K562-1043-(1-4)；18-21：9947A-1043-(1-4)；22-25：9948-1043-(1-4)
Figure 12 plasmid electrophorogram;
1:PUC18 (contrast); 2-5:K562-D21S11-(1-4); 6-9:
9947A-D21S11-(1-4)；10-13：9948-D21S11-(1-4)；14-17：
K562-CSF1PO-(1-4)；18-21：9947A-CSF1PO-(1-4)；22-23：
9948-CSF1PO-(1-4)；
Figure 13 is reorganization plasmid enzyme restriction electrophorogram;
1:pUC18; 2: recombinant plasmid D3S1358; 3-25: recombinant plasmid is cut with restriction enzyme ScaI enzyme; 3:Amelogenin-X; 4:Amelogenin-Y; 5-8:D3S1358 (14R, 15R, 16R, 17R); 9-11:D5S818 (11R, 12R, 13R); 12-15:D6S1043 (11R, 12R, 15R, 18R); 16-18:D7S820 (9R, 10R, 11R); 19-20:D8S1179 (12R, 13R); 21-25:Penta E (5R, 11R, 12R, 13R, 14R);
Figure 14 is reorganization plasmid enzyme restriction electrophorogram;
1:pUC18; 2-5: recombinant plasmid D12S391, D13S317, D16S539, D18S51;
6-25: recombinant plasmid is cut with restriction enzyme ScaI enzyme; 6-9:D12S391 (18R, 20R, 23R, 24R); 10-11:D13S317 (8R, 11R); 12-13:D16S539 (11R, 12R); 14-17:D18S51 (15R, 16R, 18R, 19R); 18-20:Th01 (6R, 8R, 9.3R); 21-22:Tpox (8R, 9R); 23-25:vWA (16R, 17R, 18R);
Figure 15 is reorganization plasmid enzyme restriction electrophorogram;
1: recombinant plasmid D19S433; 2-9: recombinant plasmid is cut with restriction enzyme ScaI enzyme;
2-5：D19S433(18R、20R、23R、24R)；6-9：FGA(21R、23R、24R、26R)；
Figure 16 is reorganization plasmid enzyme restriction electrophorogram;
1: recombinant plasmid D2S1338; 2-7: recombinant plasmid is cut with restriction enzyme A at II enzyme; 2-4:D2S1338 (17R, 19R, 23R); 6-9:D21S11 (29R, 30R, 31R)
Figure 17 is reorganization plasmid enzyme restriction electrophorogram;
1-2: recombinant plasmid Penta D, CSFlPO; 3-10: recombinant plasmid is cut with restriction enzyme A at II enzyme; 3-6:Penta D (8R, 9R, 12R, 13R); 7-10:CSFlPO (9R, 10R, 11R, 12R).
Embodiment
The invention discloses a kind of human DNA STR and detect the preparation method who uses reference material, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Method of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
The present invention has chosen 19 str locus seats (CSFlPO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta D, Penta E, Th01, Tpox, vWA) and sex marker site Amelogenin prepares STR somatotype reference material; With human genome K562,9947A and 9948 as template, synthetic or obtain the gene fragment that contains 19 str locus seats and sex marker site Amelogenin that the present invention chooses with polymerase chain reaction (PCR) amplification, the said gene fragment is inserted into the multiple clone site of plasmid pUC18, be built into recombinant plasmid, recombinant plasmid is converted into again to be cultivated among the competence bacterium JM109 and breeding, filter out to contain by order-checking then and insert correct clone and the genotype of fragment, and preserve bacterium liquid; Choose the bacterium liquid of these preservations, incubated overnight, extract recombinant plasmid, with the recombinant plasmid that extracts, selection is away from the carrier multiple clone site and have only the Restriction Enzyme single endonuclease digestion recombinant plasmid of a restriction enzyme site in recombinant plasmid, make recombinant plasmid be wire, contain locus D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, FGA, Penta E, Th01, Tpox, the recombinant plasmid of vWA and sex marker site Amelogenin is cut with Restriction Enzyme Sca I enzyme, contain locus PentaD, D21S11, the recombinant plasmid of CSFlPO and D2S1338 is cut with Restriction Enzyme Aat II enzyme, and enzyme is cut the back purifying.The recombinant plasmid that above-mentioned purifying is good with the Restriction Enzyme single endonuclease digestion, the genotype that comprises according to 20 locus among the 1ng/ μ L human genome K562,9947,9948 and contain this genotypic molar weight, the different genotypic plasmid equal proportions that contains is mixed, be configured to 3 cover forensic dna reference materials.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1: design of primers
Select suitable str locus seat.Mainly use the AmpFISTR@Sinofiler of ABI company according to present China
TM, the PowerPlex16 of Promega company and the development of China Material Evidence Identification Center, Ministry of Public Security DNATyper15
TMUsed str locus seat in the commercial kit, 19 str locus seats (CSFlPO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta D, Penta E, Th01, Tpox, vWA) in the special option table 1 of the inventor and sex marker site Amelogenin prepare STR detection reference material.
Design the primer of above-mentioned 19 str locus seats and sex marker site Amelogenin.Above-mentioned 19 the str locus seats and the sex marker site Amelogenin complete sequence that find from GenBank and pertinent literature, it is several to primer to be with the sequence that finds that template is passed through Primer Premier software design, filters out locus of a pair of primer amplification with Oligo software then.When the primer of design STR somatotype reference material, must guarantee that tumor-necrosis factor glycoproteins in the locus is positioned at the mid-way of amplified fragments, and apart from forward and reverse primer all between 400-600bp.Because two the site homologys of X, Y among the sex marker site Amelogenin are not very high, therefore need respectively design a pair of primer at two sites of X, Y.The primer of design adds restriction enzyme site at 5 ' end, helps the PCR product is inserted in the plasmid pUC18.The primer sequence of design sees Table 1.
The primer sequence of table 1. design
Annotate: underscore is the restriction enzyme site sequence
Embodiment 2:
Then, respectively with human genome K562,9947A and 9948 as template, increase above-mentioned 19 str locus seats and sex marker site Amelogenin, PCR reaction system and condition be following to see Table 3 and table 4:
Table 3, pcr amplification adopt 50 μ L reaction systems
Table 4, PCR thermal circulation parameters
The amplified production agarose electrophoresis the results are shown in Figure 2-7.
Embodiment 3:
The different restriction enzyme site of primer according to design carries out double digestion with plasmid pUC18 and purified PCR product with corresponding enzyme, and the enzyme that different PCR products is used is with table 5.
Used enzyme when table 5, PCR product double digestion
Plasmid pUC18 is behind Pst I/EcoR I, Hind III/Pst I, Xba I/EcoR I, Xba I/PstI, Pst 1/BamH I, Xba I/Hind III, EcoR I/Hind III, Xba I/BamH I double digestion, and electrophoresis result is seen Fig. 8.
With the connection of spending the night of T4 ligase enzyme, change e. coli jm109 with the pUC18 behind the identical enzyme double digestion and PCR product over to, the picking transformant is identified.At first, each dull and stereotyped 4 bacterium colony of picking of going up is identified transformant with bacterium colony PCR and two kinds of methods of extraction plasmid, and qualification result is seen Fig. 9-12.Be accredited as the bacterium liquid of recon, send to order-checking behind the guarantor bacterium, every kind of genomic each locus send 4 recons order-checkings.
Embodiment 4:
19 str locus seats searching that the present invention need clone in genome K562,9947A and 9948 and the genotype of sex marker site Amelogenin see Table 6.
Table 6, clone's 19 str locus seats and sex marker site Amelogenin
Genotype in genome K562,9947 and 9948
Contain all genotypic recon order-checkings in the table 6 by analyzing sequencing result, having found, the bacterium liquid that will contain these recons is preserved.In addition, choose the bacterium liquid of these preservations, incubated overnight is extracted recombinant plasmid.By analyzing the sequence of each recon, selection is away from the carrier multiple clone site and have only the restriction enzyme single endonuclease digestion recombinant plasmid of a restriction enzyme site (Sca I or Aat II) in recombinant plasmid, promptly contain locus D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, FGA, Penta E, Th01, Tpox, the recombinant plasmid of vWA and sex marker site Amelogenin is cut with restriction enzyme Sca I enzyme, contain locus Penta D, D21S11, the recombinant plasmid of CSFlPO and D2S1338 is cut with restriction enzyme A at II enzyme, make recombinant plasmid be wire, enzyme is cut the back purifying, and recombinant plasmid single endonuclease digestion purifying rear electrophoresis the results are shown in Figure 13-17.
The homogeneity (PCR output is identical) of PCR product when the plasmid single endonuclease digestion helps others and is template PCR with recombinant plasmid, if single endonuclease digestion not, plasmid is superhelix, ring-type and three kinds of forms of wire, and these three kinds of form ratios be can not determine, Taq is not easy to combine with superhelix, cyclic plasmid in the PCR process, causing with three kinds of form plasmids is that template PCR efficient is inconsistent, thereby influences the homogeneity of PCR product.Behind the single endonuclease digestion plasmid, plasmid all is wire, PCR efficient unanimity.
The recombinant plasmid that above-mentioned purifying is good with the Restriction Enzyme single endonuclease digestion, the genotype that comprises according to 20 locus among the 1ng/ μ L human genome K562,9947,9948 and contain this genotypic molar weight, the different genotypic plasmid equal proportions that contains is mixed, and being configured to the detection of human DNA STR is 3 cover forensic dna reference material K562,9947A, 9948 analogues with reference material.
Embodiment 5: 3 cover forensic dna reference material checkings of the method for the invention preparation
The employing commercial kit "
PCR AmplificationKit " detect embodiment of the invention 1-4 and be configured to 3 cover forensic dna reference material K562,9947A, 9948 analogues and human genome K562,9947A, 9948 difference.
Concrete experimental procedure: detect human genome 9947A, 9948, K562 and each genomic genotype of their analogues with commercial kit (as the AmpFISTR@SinofilerTM of ABI company).
Interpretation of result: whether the genotype of each locus is identical in human genome 9947A and the DNA reference material 9947A analogue detected result, if identical, illustrate that the DNA reference material 9947A analogue that we prepare can replace human genome 9947A as reference material; If inequality, then just can not replace human genome 9947A as reference material.Experimental result proves, the genotype of each locus is identical in the detection of human genome 9947A and DNA reference material 9947A analogue of the present invention, human genome 9948 and DNA reference material 9948 analogues of the present invention, human genome K562 and DNA reference material K562 analogue of the present invention, figure summary as a result.Can replace human genome 9947A, 9948, K562 respectively as reference material with DNA reference material 9947A, 9948, the K562 analogue of the present invention's preparation.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. a method for preparing human DNA STR detection with reference material is characterized in that, may further comprise the steps:
Step 1, with human genome as template, synthetic or pcr amplification obtain to contain the target gene fragment of human DNA STR locus;
Step 2, with the target gene fragment of step 1 and plasmid vector reorganization, recombinant plasmid is transferred in the cell, filters out the positive colony that contains target gene fragment and the evaluation of checking order;
Recombinant plasmid in the positive colony that step 3, extraction step 2 are identified, the recombinant plasmid equal proportion that will contain different genotype is mixed, and the DNA STR that is configured to human genome detects and uses reference material;
Described target gene fragment contains the fragment of str locus seat CSFlPO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta D, Penta E, Th01, Tpox, vWA and sex marker site Amelogenin respectively, carries out pcr amplification respectively with primer sequence shown in the SEQ ID No.1-SEQ ID No.42.
2. method according to claim 1, the described cell of step 2 is a Bacillus coli cells, described plasmid vector is the corresponding plasmids of intestinal bacteria.
3. method according to claim 1, the described recombinant plasmid of step 3 is a linear plasmid.
4. the test kit that contains the DNA STR reference material of claim 1 preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101858340A CN102251032B (en) | 2011-07-01 | 2011-07-01 | Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101858340A CN102251032B (en) | 2011-07-01 | 2011-07-01 | Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102251032A CN102251032A (en) | 2011-11-23 |
| CN102251032B true CN102251032B (en) | 2013-07-24 |
Family
ID=44978600
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101858340A Active CN102251032B (en) | 2011-07-01 | 2011-07-01 | Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102251032B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105602940A (en) * | 2016-01-29 | 2016-05-25 | 公安部第一研究所 | Method for preparing human gene STR (short tandem repeat) sequence allelic ladder by using site-directed mutagenesis technology |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286477B (en) * | 2011-07-12 | 2013-06-05 | 公安部物证鉴定中心 | Method for preparing short tandem repeat (STR) parting standard substances |
| 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 |
| CN106119359A (en) * | 2016-06-29 | 2016-11-16 | 公安部物证鉴定中心 | A kind of STR typing positive control |
| 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 |
| CN109266758A (en) * | 2018-08-21 | 2019-01-25 | 广州吉妮欧生物科技有限公司 | A kind of method of cell STR identification 9 site, one step detection |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312371A (en) * | 2001-03-05 | 2001-09-12 | 四川大学 | Molecular cloning prepn of short tandom human gene repeated sequence typing reference material |
| CN1446921A (en) * | 2002-03-25 | 2003-10-08 | 江斌 | New technique for preparing STR allelomorphic gene stairs and kit possessing the stairs |
| CN101956008A (en) * | 2010-09-10 | 2011-01-26 | 无锡中德美联生物技术有限公司 | Rapid compound PCR (Polymerase Chain Reaction) amplification fluorescence assay kit for 16 gene loca |
-
2011
- 2011-07-01 CN CN2011101858340A patent/CN102251032B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1312371A (en) * | 2001-03-05 | 2001-09-12 | 四川大学 | Molecular cloning prepn of short tandom human gene repeated sequence typing reference material |
| CN1446921A (en) * | 2002-03-25 | 2003-10-08 | 江斌 | New technique for preparing STR allelomorphic gene stairs and kit possessing the stairs |
| CN101956008A (en) * | 2010-09-10 | 2011-01-26 | 无锡中德美联生物技术有限公司 | Rapid compound PCR (Polymerase Chain Reaction) amplification fluorescence assay kit for 16 gene loca |
Non-Patent Citations (2)
| Title |
|---|
| 用克隆技术制备STR等位基因;袁丽等;《中国人民公安大学学报(自然科学版)》;20070930(第3期);第41页左栏1.2方法 * |
| 袁丽等.用克隆技术制备STR等位基因.《中国人民公安大学学报(自然科学版)》.2007,(第3期), |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105602940A (en) * | 2016-01-29 | 2016-05-25 | 公安部第一研究所 | Method for preparing human gene STR (short tandem repeat) sequence allelic ladder by using site-directed mutagenesis technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102251032A (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102251032B (en) | Preparation method of reference material (RM) for detecting human DNA (deoxyribonucleic acid) STR (short tandem repeat) | |
| Kedes et al. | The organization of sea urchin histone genes | |
| Brown et al. | Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21 | |
| CN109880911A (en) | The composite amplification reagent kit of 25 human chromosomal locus and its application | |
| CN107904317B (en) | Human autosomal STR polymorphic site composite amplification kit and application thereof | |
| CN112481417B (en) | Rapid detection method and kit for new coronavirus typing and mutation sites | |
| Cellamare et al. | New insights into centromere organization and evolution from the white-cheeked gibbon and marmoset | |
| Jesionek et al. | Fundamentally different repetitive element composition of sex chromosomes in Rumex acetosa | |
| CN111826426A (en) | Method for detecting molecular marker based on KASP technology | |
| CN112322733A (en) | Nucleic acid composition and kit for detecting EGFR gene mutation and method for detecting EGFR gene mutation | |
| McKinney et al. | Members of the Arabidopsis actin gene family are widely dispersed in the genome | |
| CN102286476B (en) | Short tandem repeat (STR) parting standard substance | |
| CN104031989B (en) | The test kit of the composite amplification of a kind of human gene group DNA 26 locus | |
| Wang et al. | Survey of major capsid genes (g23) of T4-type bacteriophages in Japanese paddy field soils | |
| CN108753952A (en) | A kind of gene parting detecting reagent for 10 common mutations sites of mankind SLC25A13 genes | |
| CN109022594B (en) | Cattle AHSG gene SNP marker related to conversion efficiency of beef cattle feed | |
| Katula et al. | Ontogenic expression of a Cyl actin fusion gene injected into sea urchin eggs | |
| CN102286477B (en) | Method for preparing short tandem repeat (STR) parting standard substances | |
| CN114836520A (en) | Method for preparing short tandem repeat sequence typing reference substance | |
| CN108060212B (en) | DNA typing identification kit | |
| Xin et al. | Population genetic analysis of 6 Y-STR loci in Chinese northwestern Qinchuan yellow cattle breed | |
| CN108642190B (en) | Forensic medicine composite detection kit based on 14 autosomal SNP genetic markers | |
| Southern | DNA sequences and chromosome structure | |
| Fang et al. | STR profiling of human cell lines: challenges and possible solutions to the growing problem | |
| CN101691607B (en) | Method and reagent kit for testing human gene typing by labeling mini-STR (short tandem repeats) with fluorescence |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 100038 Beijing city Xicheng District Muxidi No. 17 South Lane Patentee after: Material Evidence Identification Center, Ministry of Public Security Patentee after: SUZHOU NUHIGH BIOTECHNOLOGY CO., LTD. Address before: 100038 Beijing city Xicheng District Muxidi No. 17 South Lane Patentee before: Material Evidence Identification Center, Ministry of Public Security Patentee before: Suzhou NuHigh Biotechnology Co., Ltd. |