CN104745691A - Primer group complexly amplified by fluorescent mark for synchronously analyzing 27 genetic locus of human genomic DNA, kit and application - Google Patents
Primer group complexly amplified by fluorescent mark for synchronously analyzing 27 genetic locus of human genomic DNA, kit and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention relates to a primer group for fluorescence labeling compound-amplification used for synchronously analyzing 27 genetic loci of human genomic DNA, a kit and application thereof the kit and belongs to the technical field of molecular biology. 27 genetic loci are divided into five groups and totally relate to fluorescence labels of six colors; a fluorescence labeling compound amplification system disclosed by the invention is high in sensitivity and capable of detecting all 27 genetic loci when the DNA template amount is 0.12ng. The system is suitable for directly amplifying blood filter paper and FTA card acquired samples, and can be applied to individual recognition and paternity test.
Description
Technical field
The primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus, test kit and application while of the present invention relates to a kind of, belong to technical field of molecular biology, this system can be applied to individual recognition and paternity test.
Background technology
Tandem repeat loci (Short Tandem Repeat, STR) is the genetic marker generally applied at present.STR mark is compared with other genetic markers, str locus seat fragment is little, easily increase, be more suitable for trace and degraded sample, the amplification condition of each locus is close and can composite amplification, thus have quick, efficient, accurate, sensitive, the advantage such as contain much information.Especially setting up in DNA database, compared with the technology such as RFLP in the past, STR composite amplification technology has great superiority.Therefore U.S. FBI has carried out large quantity research, have selected 13 str locus seats for setting up DNA database---CODIS (Combined DNA Index System): CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, vWA, these str locus seats are commonly called 13 core gene seats.
Just because of the application prospect that STR possesses, method medical circles and some companies have all carried out large-scale exploration research to it.The middle and later periods nineties is with American AB I (Applied Biosystems, USA) for the poly-talented company of representative sets up composite amplification and fluorescent detection system, and the most that has been born representational be the PowerPlex-16 fluorescence detection reagent kit of IdentifilerTM and Promega (USA) company of ABI company.These two test kits include above-mentioned 13 core gene seats, are respectively 5 looks and 4 color reagent boxes.
Long-term DNA inspection practice shows, the genetic polymorphism of str locus seat exists certain difference between race, and between each locus, difference is also very large.In 13 core gene seats that U.S. FBI recommends, there is portion gene seat genetic polymorphism not high, or differ greatly between different crowd, and be not exclusively applicable to Chinese population, certain impact is brought to the application of DNA inspection technology, therefore, increase the detection locus number be applicable to, thus increase resolving power, improve detection efficiency, it is a large requirement of STR Fluorescence kit development, propose 13CODIS to escalate into 24 str locus seat points as Journal of Forensic Sciences FSI:Genetics in 2012 just publishes the article, except previous 13CODIS, add D2S1338, D19S433, D1S1656, D12S391, D2S441, D10S1248, Penta E, DYS391, SE33, D22S1045, 11 locus such as Penta D, additional Amelogenin, amount to 25 sites.
What the commercial kit of main flow adopted usually in the market is five fluorescent technique technology.But along with detecting the increasing of locus number demand, multicolored fluorescence technique detects restricted the highlighting of locus number, therefore the foundation of six look detection technique of fluorescences and application, be the inexorable trend of STR fluorescence detection reagent kit.
Accordingly, we, on current widely used five fluorescent technique basis, test the 6th different look fluorescence, determine its realizability on Genetic Analyser, finally successfully establish domestic first STR six look fluorescence detecting system; The genetic polymorphism of the str locus seat of Chinese population is conducted in-depth research simultaneously, and develop the new composite amplification system comprising all upgrading CODIS sites on this basis, Y chromosome particularly for Amel loses sample, add Y chromosome long-armed on 2 locus---Y indel and DYS391 in order to differentiate, in the hope of in six look fluorescence detecting systems, thering is provided the genotyping result to 27 locus comprising Amelogenin, is the test kit that current domestic accumulative individual recognition ability and accumulation parentage exclusion probability are the highest.
Summary of the invention
The object of the invention is: the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus, test kit and application while of providing a kind of, detected by six look fluorescent detection systems simultaneously, carry out the STR compound checking system of individual recognition and paternity test.Relate to the STR genetic marker detecting and there is in human genome polymorphism.The present invention be more particularly directed to increase in an individual system multiple short tandem repeat with polymerase chain reaction simultaneously.
Technical scheme:
While analyst's genomic dna 27 locus the primer sets of fluorescence labeling composite amplification, include the primer pair for following 27 locus that increase: D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E, TPOX, TH01, D2S1338, CSF1PO, Penta D, D10S1248, D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA, D22S1045, Y indel, D2S441, DYS391, SE33 and Amelogenin.
The sequence of above-mentioned primer pair and the concentration in amplification system as shown in table 1:
The each locus primer sequence of table 1 and concentration
5 ' of primer end is had at least to carry out fluorochrome label in described each primer pair.
Described primer pair is through packet marking.
D3S1358, D13S317, D7S820, D16S539, D1S1656 and Penta E is first group;
TPOX, TH01, D2S1338, CSF1PO, Penta D and D10S1248 is second group;
D19S433, vWA, D21S11, D18S51 and D6S1043 are the 3rd group;
D8S1179, D5S818, D12S391, FGA and Amelogenin are the 4th group;
D22S1045, Yindel, D2S441, DYS391 and SE33 are the 5th group.
When often group marks be adopt in blue fluorescent dyes 6-FAM, Green fluorescent dye HEX, Yellow fluorochrome TAMRA, red fluorescence dyestuff mark ROX, purple fluorescent dyes VIG any one dye, and often not identical between group.
According to another aspect of the present invention, the detection kit including above-mentioned primer sets is additionally provided.
In described test kit, also include mark in molecular weight.
In described molecular weight, mark selects fluorescent orange SIZ.
Described test kit is by forming as follows: Reaction Mix, 10.0 μ L; Genomic dna, 0.125 ~ 5ng; Primer mixture, 5.0 μ L; The hot start Taq polymerase of 5U/ μ L, 0.5 μ L; sdH
2o, complements to 25.0 μ L; Wherein, described Reaction Mix is: .MgCl
27.5mM, Tris-HCl buffer 125mM, KCl 125mM, dNTPs 7.5m M, BSA 2mg/ml.
Beneficial effect
1, the present invention establishes six look STR fluorescence detecting systems, can improve the detection number of locus in test kit, improve the possibility of STR detection technique.
2,27 locus of the present invention are increased by the pair of primers being positioned at these locus both sides, wherein have at least 5 ' of primer end to carry out fluorochrome label in often pair of primer; System of the present invention comprises the mixture of the allelotrope standard substance corresponding to each locus, to determine the allelotrope of each locus in DNA sample; Comprise in system of the present invention for differentiating that the locus of DNA sample sex is Amelogenin locus, DYS391 and Y indel; Fluorescence labeling composite amplification system of the present invention multiple locus that increases adopts polymerase chain reaction to carry out, and detection method adopts multiple tracks or single track capillary electrophoresis.
3, the invention still further relates to a kind of method adopting this fluorescence labeling composite amplification checking system to analyze DNA sample; Wherein, the DNA sample that the present invention is suitable for derives from seminal stain, salivary stain, tissue, blood stain or blood etc.
4, in the present invention, 24 str locus seats are configured to: D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E, D2S441, TPOX, TH01, D2S1338, CSF1PO, Penta D, D10S1248, D22S1045, D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA, SE33.The present invention conducts in-depth research above-mentioned 24 tandem repeat loci and allelotrope hereditary feature thereof, shows that these 24 tandem repeat loci have genetic polymorphism and the distribution of good gene frequency of height in crowd.The present invention except Amelogenin locus, also add Y chromosome long-armed on 2 locus, in order to differentiate Y chromosome partial loss sample.Above-mentioned 24 tandem repeat loci add for differentiating other Amelogenin locus of human nature and forming composite amplification checking system of the present invention for the identification of DYS391 and the Y indel of Y chromosome.
5, the invention provides a kind of test kit being carried out individual recognition and paternity test by composite amplification 27 locus.This locus is formed, and breaches the pattern of existing 13 core gene seats, more the technical requirements of coincidence method medical science DNA inspection and the population genetics feature of Chinese population, and has taken into account ID interchange and the demand shared.
6, checking system provided by the invention reaches at present the highest level of STR fluorescence labeling composite amplification test kit in the world.
Accompanying drawing explanation
Fig. 1: to the STR genotyping result figure of DNA standard substance 9948, what show in figure is the amplification of D3S1358 isoloci;
Fig. 2: to the STR genotyping result figure of DNA standard substance 9948, what show in figure is the amplification of D19S433 isoloci;
Fig. 3: to the STR genotyping result figure of DNA standard substance 9948, what show in figure is the amplification of D22S1045 isoloci;
Fig. 4: the STR genotyping result figure of allelic ladder, what show in figure is the amplification of D3S1358 isoloci;
Fig. 5: the STR genotyping result figure of allelic ladder, what show in figure is the amplification of D19S433 isoloci;
Fig. 6: the STR genotyping result figure of allelic ladder, what show in figure is the amplification of D22S1045 isoloci.
Embodiment
Below by embodiment, the present invention is described in further detail.But it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (such as show with reference to J. Pehanorm Brooker etc. according to the technology described by the document in this area or condition, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1
One, the determination of locus
Carry out analysing in depth research and preferably new fine resolution locus to existing str locus seat.To D1S1656, D6S1017, D4S2408, D8S1179, D21S11, D7S820, D13S317, D16S539, TPOX, TH01, D5S818, vWA, D18S51, FGA, D2S1338, D19S433, Penta E, D10S1248, D1S1677, Penta D, CSF1PO, D3S1358, D19S253, D12S391, D6S1043, D6S474, D12ATA63, D22S1045, D11S4463, D1S1627, D3S4529, D2S441, D17S1301, D1GATA113, D18S853, D20S482, D14S1434, D5S2500, D2S1776, more than 40 the str locus seat such as D10S1435 and D9S1122 carries out investigation in the genetic polymorphism of Chinese population.Genotype detection is carried out to more than 5000 individualities, distribution frequency according to each locus allele calculates the data such as individual recognition ability (DP), heterozygosity (H), parentage exclusion probability (PE), show in 24 str locus seats, except TH01, TPOX locus, the DP value of all the other each locus is all close to 0.9, H is all greater than 0.7, PE value mostly more than 0.5, and this shows that they have good using value on legal medical expert's somatotype.Although consider that TH01, TPOX locus polymorphism is poor, polymorphism information content (PIC) is higher, and is 13CODIS locus, meets the requirement of legal medical expert's application.Selected other str locus seats PIC value of native system is also all greater than 0.5, therefore the inspection that can be biology sample provides sizable quantity of information.
Amelogenin locus once reported that its Y lost, and caused sex to be judged by accident.As a supplement, after in-depth analysis research is carried out to existing Y chromosome, select long-armed upper 2 the locus DYS391 and Y indel of Y chromosome to carry out supplementing qualification, lose sample for part male Y chromosome especially, play supplementary qualification effect; In addition DYS391 and Yindel is positioned at homochromy, has certain separating capacity to the male mixing sample of man.
Considering with on the basis of existing database compatibility, finally establish str locus seat to form, namely D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E, TPOX, TH01, D2S1338, CSF1PO, PentaD, D10S1248, D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA, D22S1045, D2S441, DYS391, SE33, separately adds Amelogenin, Yindel.This locus forms the pattern breaching existing 13 core gene seats, provides the genotyping result of 27 locus, improves accumulative individual recognition ability and the accumulation parentage exclusion probability of system, the technical requirements of coincidence method medical science DNA inspection more.
Two, the foundation of six look fluorescent detection systems
The detection technique of fluorescence that forensic laboratory adopts, has multicolor analysis, feature quick and easy to use.Its main process comprises: laser bombardment is connected to the fluorophor (dyestuff) of DNA end, fluorophor absorbing laser energy, then the light of more low-yield (larger wavelength) is sent, the utilizing emitted light of particular range of wavelengths is collected with spectral filter, the signal of fluorophor is come from order to collection and amplification with photomultiplier or electric coupling device, and be translated into electrical signal, the final peak figure generated in capillary electrophoresis.Namely multicolor fluorescence detection technique refers to different fluorescein-labelled primers, detects respectively at each wavelength, thus reaches the object detecting composite amplification product.At the consideration physical properties of fluorescein and the instrument characteristic of Genetic Analyser, select the new fluorescein of suitable wavelength; Mix with existing multicolored fluorescent mark, form six new look detection system, set up new fluorophor resolution algorithm (matrix) simultaneously.
Three, the locus assembled scheme design of fluorescence labeling composite amplification system
This research has been carried out discriminating to fluorescence dye, has been selected, and has selected blue, green, yellow, red, purple, orange six kinds of fluorescent markers, preferred 6 look fluorescence assembled schemes.
On the basis determining 6 look fluorescence assembled schemes, by repeatedly testing in a large number, be below wherein a kind of product mix mode: D3S1358, D13S317, D7S820, D16S539, D1S1656 and Penta E is first group, the fluorescent marker of the primer that this group locus is corresponding is any one of 6-FAM, HEX, TEMRA and ROX; TPOX, TH01, D2S1338, CSF1PO, Penta D and D10S1248 is second group, and the fluorescent marker of the primer that this group locus is corresponding is and a different set of fluorochrome label thing; D19S433, vWA, D21S11, D18S51 and D6S1043 are the 3rd group, and the fluorescent marker of the primer that this group locus is corresponding is the fluorochrome label thing all different with second group from first group; Gender-specific genes seat Amelogenin, D8S1179, D5S818, D12S391, FGA are the 4th group, and the fluorescent marker of the primer that this group locus is corresponding is the fluorochrome label thing all different with the 3rd group from first group, second group; D22S1045, Yindel, D2S441, DYS391, SE33 are the 5th group, and the fluorescent marker of the primer that this group locus is corresponding is and first group, second group, the 3rd group fluorochrome label thing all different with the 4th group; Interior mark selects fluorescent orange to mark, and fluorescent marker is SIZ.Detect while the locus array mode of this original creation makes only to need mark five kinds of fluorescence just can realize these 27 locus and analyze.
Four, the optimization of florescence labeling STR multiplex system and foundation
1, the allotment of quality of balance between locus
Along with the increase of locus number in composite amplification system, due to the impact of competition, the relative equilibrium of each locus controls difficulty and strengthens, and by repeatedly repeatedly testing, regulating primer concentration and proportioning, reaching balance eventually.Each locus primer sequence and primer concentration as shown in table 1.With the primer concentration in form, be configured to multiple expansion primer mixture.Primer synthesis and mark ABI 394 synthesizer complete.
2, the foundation of composite amplification condition
First the single amplification condition of 27 locus is optimized, on the basis successfully establishing individual gene seat amplification condition, study 27 locus composite amplification PCR reaction conditionss, the parameters determined in composite amplification is repeatedly tested by a large amount of, as, the change of loop parameter, annealing temperature, damping fluid ionic strength, enzyme amount, composite amplification reaction volume and template DNA amount etc., amplified production is made to reach balance, special requirement, set up composite amplification system, amplify 27 locus simultaneously.
Final amplification step is as follows:
A, amplification system are:
Table 2 amplification system
| Component | Volume |
| Reaction Mix | 10.0μL |
| Genomic dna | 1 ~ 5 μ l, content is 0.125 ~ 5ng |
| As above the primer that 27 locus are corresponding | 5.0μL |
| Hot start Taq polymerase (5U/ μ L) | 0.5μL |
| sdH 2O | Complement to 25.0 μ L |
B, amplification thermal cycling
L pcr amplification pipe is placed on thermal cycler by ();
(2) program of recommending is selected to increase below;
(3) sample after amplification should keep in Dark Place;
The amplification program of table 3 thermal cycler
The fluoroscopic examination on genetic analyzer of C, amplified production
Loading mixture ((0.5 μ l AGCU Marker SIZ-500 (Zhongde Meilian Biotech Co., Ltd. Wuxi)) × (sample introduction number)+(12 μ l deionized formamide) × (sample introduction number)) is formed by marking AGCU Marker SIZ-500 in deionized formamide and system middle-molecular-weihydroxyethyl.12.5 μ l loading mixtures are mixed with 1 μ l amplified production or system allelic analytical standard EX27 Allelic Ladder (Zhongde Meilian Biotech Co., Ltd. Wuxi), avoids producing bubble.95 DEG C of sex change 3 minutes, ice bath 3 minutes, and electrophoresis as early as possible; Detect with genetic analyzer and analyze;
D, phenotypic analysis
The data of collecting are detected with genetic analyzer in fragment analysis software GeneMapper analytical procedure 3, sample analysis data and EX27 Allelic Ladder somatotype standard substance (see Fig. 4 ~ 6) are compared, obtain the typing data of actual sample DNA standard substance 9948, the results are shown in Figure 1 ~ 3.
On the basis of assembled scheme, the flanking sequence according to 27 locus positions carries out design of primers, realizes the composite amplification of 27 locus in same reaction.The present invention sets up six look fluorescence detecting systems at home first, and realizes the composite amplification of 27 locus simultaneously in single reaction.
The fluorescence labeling composite amplification checking system that embodiment 2 applies 27 locus carries out triplet paternity test
1, the blood sample in paternity test case is collected: in this experiment, sample is provided by XX paternity test mechanism.DNA extraction adopts Chelex-100 method to extract the genomic dna of 3 whole blood samples respectively: get the 1.5ml centrifuge tube that 0.5 ~ 5 μ l whole blood is placed in sterilizing, add sdH2O 1ml in pipe, the vibration several seconds; Be placed in room temperature after 10 minutes, the vibration several seconds, centrifugal 3 minutes of 12,000rpm, abandons supernatant liquor, retains enough supernatant liquor drowning precipitation, does not stir precipitation; Add the Chelex-100 of 200 μ l 5%, the vibration several seconds; In 56 DEG C of insulations 30 minutes, the vibration several seconds; Boiling water bath 10 minutes, the vibration several seconds; Centrifugal 5 minutes of 12,000rpm, supernatant is extract the genomic dna obtained.The extraction of genomic dna is carried out with reference to " GA/T 383-2002 forensic DNA profiling laboratory inspection specification ".Also directly with blood examination, can be carried out by 1.2mm aperture blood filter paper or FTA card blood sample sheet.
2, augmentation detection
Carry out pcr amplification according to embodiment 1, genetic analyzer detects and finally obtains genotyping result.
3, conclusion
Result display (see table 4), tested father, mother, female all meet genetic development in 23 locus detected, and calculate relative parentage possibility (RCP) and are greater than 99.999999%, can assert parent child relationship.
The detected result of table 4 AGCU EX27
The ultimate principle of paternity test is: according to mendel's law, and parental gene type determines progeny genotypes.Under the prerequisite not having transgenation, somatotype mistake: 1. the pair of alleles of child must be one from father, one from mother; 2. the allelotrope that all can have with parents of child.
PI＝X/Y＝∑f×c/∑f×p
F represents that breeder mother is to the required allelotrope chance of child;
C represents that father is to the required allelotrope chance of child;
P represents that random man is to the required allelotrope chance of child, equals required gene frequency;
Relative parentage possibility (RCP)=(CPI/ (CPI+1)) × 100%; Wherein CPI is the product of each not linked gene seat PI;
According to above-mentioned calculating, in this experiment, father-female-female triplet paternity test RCP value is for being greater than 99.999999%, assert parent child relationship.
Sequence table
<110> Zhongde Meilian Biotech Co., Ltd. Wuxi
<120> mono-kind is the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus, test kit and application simultaneously
<130>
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<170> PatentIn version 3.5
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<213> artificial sequence
<400> 36
gctagtctcg aatttgaccc tt 22
<210> 37
<211> 20
<212> DNA
<213> artificial sequence
<400> 37
ccacacggcc tggcaactta 20
<210> 38
<211> 29
<212> DNA
<213> artificial sequence
<400> 38
tatttaccta tcctgtagat tattttcac 29
<210> 39
<211> 23
<212> DNA
<213> artificial sequence
<400> 39
tgcaagtatg tgacaagggt gat 23
<210> 40
<211> 22
<212> DNA
<213> artificial sequence
<400> 40
ctctcagagg aatgctttag tg 22
<210> 41
<211> 24
<212> DNA
<213> artificial sequence
<400> 41
gatagtagtt tcttctggtg aagg 24
<210> 42
<211> 24
<212> DNA
<213> artificial sequence
<400> 42
attcagcctc catatcactt gagc 24
<210> 43
<211> 23
<212> DNA
<213> artificial sequence
<400> 43
tccaaaagtc aaatgcccca tag 23
<210> 44
<211> 25
<212> DNA
<213> artificial sequence
<400> 44
catacttacc tccagtcgtt tcata 25
<210> 45
<211> 25
<212> DNA
<213> artificial sequence
<400> 45
agcgttcccc gatgatagta gtctc 25
<210> 46
<211> 19
<212> DNA
<213> artificial sequence
<400> 46
cgcgaatgta tgattggca 19
<210> 47
<211> 22
<212> DNA
<213> artificial sequence
<400> 47
aactctctga atcaggcaca tg 22
<210> 48
<211> 26
<212> DNA
<213> artificial sequence
<400> 48
gtttcttcaa tgctgaaagt aagtat 26
<210> 49
<211> 21
<212> DNA
<213> artificial sequence
<400> 49
gcaaggctgt aacaagggct a 21
<210> 50
<211> 21
<212> DNA
<213> artificial sequence
<400> 50
ctctccttcc caaatgttca t 21
<210> 51
<211> 27
<212> DNA
<213> artificial sequence
<400> 51
tcattcaatc atacacccat atctgtc 27
<210> 52
<211> 22
<212> DNA
<213> artificial sequence
<400> 52
tagggtgttg agcagcatct ct 22
<210> 53
<211> 22
<212> DNA
<213> artificial sequence
<400> 53
tcacgtctgt aattccagct cc 22
<210> 54
<211> 23
<212> DNA
<213> artificial sequence
<400> 54
acaacatctc tcctaccgct cta 23
Claims (10)
1. the primer sets of the simultaneously fluorescence labeling composite amplification of analyst's genomic dna 27 locus, it is characterized in that, include the primer pair for following 27 locus that increase: D3S1358, D13S317, D7S820, D16S539, D1S1656, Penta E, TPOX, TH01, D2S1338, CSF1PO, Penta D, D10S1248, D19S433, vWA, D21S11, D18S51, D6S1043, D8S1179, D5S818, D12S391, FGA, D22S1045, Y indel, D2S441, DYS391, SE33 and Amelogenin.
2. while according to claim 1, the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus, is characterized in that: the sequence of described primer pair is as follows: D3S1358 SEQ ID NO.1 ~ 2, D13S317 SEQ ID NO.3 ~ 4, D7S820 SEQ ID NO.5 ~ 6, D16S539 SEQ ID NO.7 ~ 8, D1S1656 SEQ ID NO.9 ~ 10, Penta E SEQ ID NO.11 ~ 12, TPOX SEQ ID NO.13 ~ 14, TH01 SEQ ID NO.15 ~ 16, D2S1338 SEQ ID NO.17 ~ 18, CSF1PO SEQ ID NO.19 ~ 20, Penta D SEQ ID NO.21 ~ 22, D10S1248 SEQ ID NO.23 ~ 24, D19S433 SEQ ID NO.25 ~ 26, vWA SEQ ID NO.27 ~ 28, D21S11 SEQ ID NO.29 ~ 30, D18S51 SEQ ID NO.31 ~ 32, D6S1043 SEQ ID NO.33 ~ 34, D8S1179 SEQ ID NO.37 ~ 38, D5S818 SEQ ID NO.39 ~ 40, D12S391 SEQ ID NO.41 ~ 42, FGA SEQ ID NO.43 ~ 44, D22S1045 SEQ ID NO.45 ~ 46, Y indel SEQ ID NO.47 ~ 48, D2S441 SEQ ID NO.49 ~ 50, DYS391 SEQ ID NO.51 ~ 52, SE33 SEQ ID NO.53 ~ 54 and Amelogenin SEQ ID NO.35 ~ 36.
3. the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus while according to claim 1, is characterized in that: the final concentration of described primer pair in amplification volume is as follows: SEQ ID NO.1 ~ 2,0.13 μM, SEQ ID NO.3 ~ 4,0.15 μM, SEQ ID NO.5 ~ 6,0.3 μM, SEQ ID NO.7 ~ 8,0.14 μM, SEQ ID NO.9 ~ 10,0.13 μM, SEQ ID NO.11 ~ 12,0.67 μM, SEQ ID NO.13 ~ 14,0.08 μM, SEQ ID NO.15 ~ 16,0.13 μM, SEQ ID NO.17 ~ 18,0.16 μM, SEQ ID NO.19 ~ 20,0.15 μM, SEQ ID NO.21 ~ 22,0.8 μM, SEQ ID NO.23 ~ 24,0.7 μM, SEQ ID NO.25 ~ 26,1 μM, SEQ ID NO.27 ~ 28,0.6 μM, SEQ ID NO.29 ~ 30,0.6 μM, SEQ ID NO.31 ~ 32,0.3 μM, SEQ ID NO.33 ~ 34,1 μM, SEQ ID NO.37 ~ 38,0.8 μM, SEQ ID NO.39 ~ 40,0.6 μM, SEQ ID NO.41 ~ 42,0.6 μM, SEQ ID NO.43 ~ 44,1.6 μMs, SEQ ID NO.45 ~ 46,1 μM, SEQ ID NO.47 ~ 48,0.7 μM, SEQ ID NO.49 ~ 50,0.7 μM, SEQ ID NO.51 ~ 52,0.9 μM, SEQ ID NO.53 ~ 54,2 μMs and SEQ ID NO.35 ~ 36,0.3 μM.
4. the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus while according to claim 1, is characterized in that: have at least 5 ' of primer end to carry out fluorochrome label in described each primer pair.
5. the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus while according to claim 4, is characterized in that: described primer pair is through packet marking.
6. while according to claim 5, the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus, is characterized in that: D3S1358, D13S317, D7S820, D16S539, D1S1656 and Penta E is first group; TPOX, TH01, D2S1338, CSF1PO, Penta D and D10S1248 is second group; D19S433, vWA, D21S11, D18S51 and D6S1043 are the 3rd group; D8S1179, D5S818, D12S391, FGA and Amelogenin are the 4th group; D22S1045, Yindel, D2S441, DYS391 and SE33 are the 5th group.
7. the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus while according to claim 6, it is characterized in that: when often group marks be adopt in blue fluorescent dyes 6-FAM, Green fluorescent dye HEX, Yellow fluorochrome TAMRA, red fluorescence dyestuff mark ROX, purple fluorescent dyes VIG any one dye, and often not identical between group.
8. the test kit of the primer sets of the fluorescence labeling composite amplification of analyst's genomic dna 27 locus while including described in any one of claim 1 ~ 7.
9. test kit according to claim 8, is characterized in that: in described test kit, also includes mark in molecular weight; In described molecular weight, mark selects fluorescent orange SIZ; Described test kit is by forming as follows: Reaction Mix, 10.0 μ L; Genomic dna, 0.125 ~ 5ng; Primer mixture, 5.0 μ L; The hot start Taq polymerase of 5U/ μ L, 0.5 μ L; sdH
2o, complements to 25.0 μ L; Wherein, described Reaction Mix is: .MgCl
27.5mM, Tris-HCl buffer 125mM, KCl 125mM, dNTPs 7.5m M, BSA 2mg/ml.
10. the application of test kit according to claim 8 in individual recognition and paternity test.
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| CN106591463A (en) * | 2016-12-29 | 2017-04-26 | 无锡中德美联生物技术有限公司 | Fluorescence labelled multiplex amplification primer set containing human genome DNA 22 loci, kit and application thereof |
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| CN110066790A (en) * | 2018-01-24 | 2019-07-30 | 深圳华大法医科技有限公司 | The six color fluorescence STR classifying methods and its dedicated kit of a kind of 28 gene locis of synchronous detection |
| CN110066791A (en) * | 2018-01-24 | 2019-07-30 | 深圳华大法医科技有限公司 | The six color fluorescence STR classifying methods and its dedicated kit of a kind of 21 gene locis of synchronous detection |
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