CN104946632A - Autosome STR gene locus fluorescent labeling composite amplification kit having enhanced identification ability, and applications thereof - Google Patents
Autosome STR gene locus fluorescent labeling composite amplification kit having enhanced identification ability, and applications thereof Download PDFInfo
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Abstract
The present invention discloses an autosome STR gene locus fluorescent labeling composite amplification kit having enhanced identification ability, wherein 27 STR gene loci can be simultaneously amplified with the kit and comprise 24 autosome STR gene loci such as D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D2S441, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D16S539, D22S1045, SE33 and D10S1248, 2 Y chromosome gene loci such as Y-indel and DYS391, and a sex-determining locus Amel. According to the present invention, with the application of the kit to perform the DNA gene detection, all the gene loci of the current mainstream products at home and abroad are contained, the currently existing DNA database in most countries are covered, the compatibility problem is not required to be worried about, the accumulation individual recognition rate and the combined paternity non-exclusion probability of the system are improved, and the individual discriminability is improved on the whole.
Description
Technical field
The present invention relates to a kind of pcr amplification test kit, particularly relate to the fluorescent composite amplification reagent kit that a kind of single tube detects 24 autosomal locuses, 2 Y chromosome locus, 1 sex locus simultaneously, the invention still further relates to preparation method and the application of this test kit in judicial expertise field of this fluorescent composite amplification reagent kit, belong to euchromosome somatotype and qualification field.
Background technology
STR (short tandom repeat, STR) be the DNA sequence dna as the class that core unit tandem sequence repeats is formed by 2-6 base in human genome with length polymorphism, the number of variations of its core unit and the differently composed genetic polymorphism of STR of multiplicity.STR distribution is wide, number is many, account for 10% of human genome, the quantity of information comprised is huge, different sequences can produce hundreds of millions of genotype combination, and each to be combined in the frequency occurred in colony all very low, there is high Individual identification ability, so Chang Zuowei genetic marker and be used to legal medical expert's individual recognition, Relationship iden-tification in DNA analysis technology.Along with the development of this technology, a lot of countries utilizes this technology to establish the DNA database of offender and suspect, is namely saved in database to the DNA data analysis of suspect, is convenient to compare and the job such as investigation.Also be the mainstream technology of DNA Database simultaneously.Meanwhile, the fragment of str locus seat is little, easily increases, and be suitable for inspection trace and degraded sample, and the amplification condition of each locus is similar and can meet amplification, thus have sensitive, accurate, quick, the advantage such as contain much information.Because the somatotype research of these advantage str locus seats is at home and abroad widely used in anthropology, medicogenetics and medical jurisprudence and each association area already with screening.
Have selected 13 str locus seats for setting up DNA database from U.S. FBI the earliest---CODIS (CombiTAM DNA Index System): D3S1358, TH01, D21S11, D18S51, D5S818, D13S317, D7S820, CSF1PO, vWA, D8S1179, TPOX, FGA, D16S539.These str locus seats are commonly called 13 core gene seats, and what have representative at present most is the Identifiler test kit of American AB I company and the PowerPlex-16 fluorescence detection reagent kit of Promega company, all contain 13 above-mentioned core gene seats.But along with DNA is more and more extensive as the application of identification of means, user has had more and more higher requirement to scope of application of the locus number of test kit, quantity of information, proliferation time, sample etc., and along with the continuous expansion of China DNA database establishment scale, the effect of comparing also becomes more and more important, because comparing is based upon on identical str locus seat, therefore also need STR test kit to have compatibility on locus, otherwise the data resource of DNA lane database part can be caused to waste.
The fluorescence labeling composite amplification system of multiple locus of analyst's genomic dna while that patent CN101144774 (mankind STRTYPER pcr amplification fluorescence detection reagent kit) disclosing a kind of, for detecting following 21 autosomal locuses: TPOX, D3S1358, D5S818, FGA, CSF1PO, D8S1179, D7S820, TH01, VWA, D13S317, D16S539, D18S51, D21S11, D12S391, D19S433, D1S1656, D2S1338, D6S1043, PentaE, PentaD, Amel.
In order to reduce detection time, improve the individual recognition ability of test kit and accumulative parentage exclusion probability, can the DNA database of most countries covering the whole world, and save reagent and human cost simultaneously, increase work efficiency.Need to research and develop a kind of euchromosome STR locus fluorescence labeling composite amplification test kit with enhancing distinguishing ability newly.
Summary of the invention
Technical problem to be solved by this invention there is provided a kind of euchromosome STR locus fluorescence labeling composite amplification test kit with enhancing distinguishing ability, contain the full gene seat that each manufacturer domestic and international at present adopts, for forensic identification and paternity test.This test kit is passing in CN101144774 (STRtyper-21G) the insertion and deletion fragment Y-indel on a Y chromosome locus DYS391 and Y chromosome that with the addition of conventional SE33, D2S441, D10S1248, D22S1045 tetra-autosomal locuses in Europe and auxiliary sex identification locus Amel under 21 locus, can be increased 27 locus simultaneously in primary first-order equation, database investigation is reached great fraction of coverage, save the cost of reagent and manpower greatly, improve working efficiency simultaneously.
Described locus is D3S1358, TH01, D21S11, D18S51, Penta E, Y-indel, DYS391, D12S391, D6S1043, D2S1338, D1S1656, Amel, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, FGA, D16S539, D22S1045, SE33 and D10S1248.
Amplification system of the present invention comprises primer mixture, reaction buffer etc.
First specific primer is designed for above-mentioned 27 locus respectively at the flank of its tumor-necrosis factor glycoproteins.Design of primers adopts Primer5 software, and every bar primer annealing temperature is at about 60 DEG C.Can not produce primer dimer, other interacts or cross reaction, amplified production length is between 70-500bp.Amplification assay is carried out to often pair of primer and optimizes, until obtain clear single amplified band.Primer sequence sees the following form 1.
The corresponding primer sequence of table 1, each locus
The present invention is by the Rational Arrangement of each locus and the high fluorescence dye of preferred a series of fluorescence intensity carries out mark packets in often pair of primer, be respectively first group of FAM mark: D3S1358, TH01, D21S11, D18S51 and Penta E, second group of HEX mark: Y-indel, DYS391, D12S391, D6S1043, D2S1338 and D1S1656, 3rd group of TAM mark: D5S818, D13S317, D7S820, D19S433, CSF1PO and Penta D, 4th group of ROX mark: D2S441, vWA, D8S1179, TPOX and FGA, 5th group of Alex 594 marks: Amel, D16S539, D22S1045, SE33 and D10S1248, interior mark selects fluorescent orange to mark, fluorescent marker is Atto 633.Often by the fragment length of primer amplification, each locus is separated among group, and optimize primer sequence and make there is not non-specific band within the scope of amplification, rear adjustment primer concentration makes the peak height harmony between a same fluorescein different genes seat reach 50%, average peak between different fluorescein is up to 30%, wherein harmonious requirement is not done to the locus on Y-indel and DYS391 two Y chromosomes, but need can carry out somatotype accurately under positive control 0.125ng.
The method that the present invention detects amplified production measures for adopting multiple tracks or single track capillary electrophoresis genetic analyzer; The template that the present invention measures comprises the blood of people, blood stain, seminal fluid, saliva, body fluid, hair, muscle or histoorgan, and the samples such as filter paper, FTA card, cotton-wool, buccal swab that can directly increase.
There is the euchromosome STR locus fluorescence labeling composite amplification test kit strengthening distinguishing ability, comprise seedless sour water, PCR Master Mix, Primer Mix, Allelic ladder mixture, interior mark size-500.It is worth mentioning that PCR Master Mix of the present invention makes this product compatible all common sample types on the market can comprise whatman FTA card through a series of optimization experiment, whatman saliva is blocked, blood filter paper, rich female FTA card, rich female saliva card, hair, mouth desquamated cells, extract the various samples such as DNA, this test kit even external at home is also not yet accomplished, in addition, damping fluid after this kind of improvement can improve amplification efficiency greatly, the time of effective shortening product terminal adenosine acidylate, shorten overall proliferation time, and the amplification efficiency of long segment can be improved, improve the harmony of product.Its main component has: DMSO, Tris-buffer, Repone K, ammonium sulfate and dNTP etc.
The enzyme that amplified reaction needs is warm start archaeal dna polymerase, and antibody modification or chemically modified, need the enzyme adding 2U-4U in the system of this reaction.
Amplification system (as ABI9700, ABI9600, Bio-Rad S1000 etc.) on various reaction heat circulating instrument adopts following program can obtain good result: 95 DEG C of insulation 2-10 minute; 94 DEG C are incubated 5-10 second, and 61 DEG C were incubated for 60 seconds, and 70 DEG C are incubated 30-60 second, and this step runs 28 circulations; 60 DEG C of insulation 15-30min, 4 DEG C of insulations.
The present invention is owing to have employed fluorescently-labeled primer, amplified production is also with fluorescent marker, and marker can send the optical signal that can be identified by genetic analyzer (as ABI3500,3500genetic analyzer) under laser excitation, so amplified production can by the instruments such as genetic analyzer carrying out electrophoresis and detect analyzing.
In genetic analyzer detects, amplified production marks (SIZE-500) with in molecular weight, methane amide mixes according to a certain percentage, enters electrophoretic separation in instrument kapillary or gel.Mark is made up of the fluorescent label DNA fragment of many known length in molecular weight, is used for calculating pcr amplification product fragment length, thus can judge gene type and with allelic ladder comparison.
Data after electrophoresis can be analyzed in GeneMapper ID-X data analysis software, obtain str locus somatotype collection of illustrative plates and data.
Beneficial effect of the present invention:
The present invention is passing on the insertion and deletion fragment Y-indel on a Y chromosome locus DYS391 and Y chromosome that with the addition of conventional SE33, D2S441, D10S1248, D22S1045 tetra-autosomal locuses in Europe and auxiliary sex identification locus Amel under CN101144774 (STRtyper-21G) 21 locus, can be increased 27 locus simultaneously in primary first-order equation, database investigation is reached great fraction of coverage, save the cost of reagent and manpower greatly, improve working efficiency simultaneously.
The present invention is after different fluoresceins has been tried out in screening, and have selected the fluorescence technique of six looks, the multicolored fluorescence technique more generally popular than market has had the breakthrough of matter.The detection site quantity of test kit provided by the present invention more than like product both domestic and external on the market, thus greatly improves cumulative individual insight and the accumulation parentage exclusion probability of system, improves individual resolving ability generally.Two Y chromosome locus energy auxiliary sex sites of simultaneously adding, especially when male Y chromosome gender-specific genes lacks, for Sex estimation, make test kit practicality and functionally to increase.Following table 2 couples of the present invention and domestic market limited loci main flow test kit locus information compare:
Table 2: the present invention (SureID PanGlobal) compares with domestic market limited loci main flow test kit locus information
Note :+representing that this locus comprises ,-expression does not comprise
Accompanying drawing explanation
Fig. 1 Control DNA 9948 sample graph
Fig. 2 allelic gene typing standard map.
Fig. 3-a tested father's somatotype collection of illustrative plates.
The somatotype collection of illustrative plates of Fig. 3-b child.
The somatotype collection of illustrative plates that Fig. 4 male Y chromosome gender-specific genes disappearance sample increases with test kit of the present invention.
The somatotype collection of illustrative plates that Fig. 5 male Y chromosome gender-specific genes disappearance sample increases with PowerPlex 18D test kit.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
The determination of specific embodiment one locus
The basis of the last product of our company with the addition of the SE33 that Europe is conventional, D2S441, D10S1248, insertion and deletion fragment Y-indel on a Y chromosome locus DYS391 and Y chromosome of D22S1045 tetra-autosomal locuses and auxiliary sex identification locus Amel, total total D3S1358, TH01, D21S11, D18S51, Penta E, Y-indel, DYS391, D12S391, D6S1043, D2S1338, D1S1656, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, FGA, Amel, D16S539, D22S1045, SE33 and D10S1248 be totally 27 locus.
Specific embodiment two fluorescent mark meets the locus assembled scheme design of amplification system
The present invention has carried out discriminating to fluorescence dye, has selected, and has selected blue, green, yellow, red, purple, orange six kinds of fluorescent markers, has constructed 6 look fluorescence assembled schemes.On the basis determining 6 look fluorescence assembled schemes, by repeatedly testing in a large number, design locus array mode and fluorescent mark type.From the viewpoint of production cost and each locus primer amplification efficiency etc., 27 locus are divided into 5 groups, use FAM, HEX, TAM, ROX and Alex 594 packet marking, in molecular weight, the mark fluorescence dye Atto 633 that the 6th kind of color is orange marks.A kind of preferred fluorochrome label is finally determined through screening, first group of FAM mark: D3S1358, TH01, D21S11, D18S51 and Penta E, second group of HEX mark: Y-indel, DYS391, D12S391, D6S1043, D2S1338 and D1S1656, 3rd group of TAM mark: D5S818, D13S317, D7S820, D19S433, CSF1PO and Penta D, 4th group of ROX mark: D2S441, vWA, D8S1179, TPOX and FGA, 5th group of Alex 594 marks: Amel, D16S539, D22S1045, SE33 and D10S1248, interior mark selects fluorescent orange to mark, fluorescent marker is Atto 633.This locus array mode makes only to need mark 6 kinds of fluorescence just can realize 27 locus and detects analysis simultaneously.
Specific embodiment three
The present invention has the euchromosome STR locus fluorescence labeling composite amplification test kit strengthening distinguishing ability, and this test kit comprises:
1)PCR Master Mix
2)Primer Mix
3)Control DNA 9948A
4) Allelic Ladder allelic gene typing standard substance
5) mark in Size-500 fluorescent orange molecular weight
6) spectrum correction standard substance
Above-mentioned PCR Master Mix includes: DMSO 10mM, Tris-buffer 125mM, Repone K 125mM, ammonium sulfate 65mM, triphosphate deoxy-nucleotide (dNTPs) 7.5mM, BSA2.5mg/ml etc., can realize the common various sample in compatible amplification market.
Above-mentioned Primer Mix comprises all primers (concentration is in table 1) of amplification 27 locus, Taq enzyme 2-4U/6.25 μ l, magnesium chloride 7.5Mm etc.
Above-mentioned positive reference substance is the human genome DNA bought.
Above-mentioned Allelic Ladder allelic gene typing standard substance are each allelotrope distribution situations in some amount crowd.
In above-mentioned Size-500 fluorescent orange molecular weight, mark is a series of amplified productions for demarcating certain clip size.
Above-mentioned spectrum correction standard substance is the fluorescent PCR amplified production of 6 kinds of different size fragments.
The experimentation that specific embodiment four amplifying locus and product thereof detect
1, the configuration of reaction system
2, increase thermal cycle experiment scheme
1) pcr amplification pipe is placed on thermal cycler
2) program of recommending is selected to increase below
3) sample after amplification should keep in Dark Place
| Step | Temperature | Time |
| 1 | 95℃ | 2-10 minute |
| 2 | 94℃ | 5-10 second |
| 3 | 61℃ | 1 minute |
| 4 | 70℃ | 30-60 second |
| 5 | N/A | Repeat 2-4 step 27 time (totally 28 times) |
| 6 | 60℃ | 15-30 minute |
| 7 | 4℃ | Continue: until collect PCR primer |
3, amplified production fluoroscopic examination on genetic analyzer
Loading mixture (25-50 μ L SIZE-500+1000 μ L deionized formamide) X (sample introduction number) is formed by marking (SIZE-500) in deionized formamide and system middle-molecular-weihydroxyethyl.9 μ L loading mixtures are mixed with 1 μ L amplified production or system allelic somatotype standard substance (Allelic ladder), avoids producing bubble, as early as possible electrophoresis.Detect with genetic analyzer and analyze.
The sensitivity of specific embodiment five detection kit, specificity analyses
Sensitivity analysis: by positive control by after certain copy number doubling dilution, detect until can't detect signal through pcr amplification and capillary electrophoresis, this copy number is lowest detection line, namely the sensitivity of test kit.Maximum sensitivity can detect the DNA sample being low to moderate 0.125ng.
Specificity analyses: the inspection of fluorescence labeling composite amplification checking system pig, dog, sheep, duck, chicken, mouse, ox, the intestinal bacteria etc. of 27 locus in the present invention, do not occur specific amplification peak, show that this system has species specificity.
The application of specific embodiment six test kit provided by the present invention in paternity test
Be used for the paternity test of tested father and child's relation with test kit provided by the present invention, determination step is as follows:
1. collect the blood cake in paternity test case: paternity test sample provided by certain judicial expertise
2. the process of tested sample: the sample in this case is filter paper blood, adopts and directly increases, therefore the punch tool of 1.2mm only need be used to carry out punching as detection template
3. augmentation detection: carry out fluorescent mark, pcr amplification and genetic analyzer according to embodiment 2 ~ 5 and detect, select the test kit that specific oligonucleotide amplimer of the present invention is right simultaneously, the genotyping result of tested father is shown in Fig. 3-a, the genotyping result of child is shown in Fig. 3-b, and its comparing result sees the following form 3:
Table 3 detected result of the present invention
Note: underscore represents the locus not meeting genetic development
Conclusion:
Detect collection of illustrative plates and see Fig. 3-a/b, result display (see table 3), this boy and tested father do not meet genetic development on vWA, D12S391, SE33 tri-locus, get rid of parent child relationship.
Diad detection because parental generation one party can not provide genetic information, therefore causing the probability of its parental right to detect lower relative to triplet, so the accuracy in order to ensure paternity test, usually all needing to increase more locus in paternity test.In the paternity test of single parent's case, if find a contradiction locus, more than 26 will be increased to, if there is more than 3 or 3 contradiction locus, then can make the conclusion of " negating one's own relation ".
In this example, my department has selected PowerPlex 18D test kit to have detected 17 locus, finds that this boy does not meet genetic development on vWA locus, can not paternity excluding.Find that there is vWA and D12S391 two locus when taking charge of previous generation product STRtyper-21G with me and do not meet genetic development, still can not paternity excluding.If can increase several again by the locus of detection, significantly parentage exclusion probability can be improved.Detect 26 locus with test kit of the present invention to find, the boy of this example has 3 locus to violate genetic development simultaneously, can make the conclusion of " negating one's own " simultaneously.
The sex identification of specific embodiment seven test kit provided by the present invention under male Y chromosome gender-specific genes disappearance
By the sex identification of test kit provided by the present invention on male sex's sample Y chromosome under gender-specific genes seat disappearance, determination step is as follows:
1. collect the blood cake of male sex's sample: sample is provided by certain public security bureau
2. the process of tested sample: the sample in this case is filter paper blood, adopts and directly increases, therefore the punch tool of 1.2mm only need be used to carry out punching as detection template
3. augmentation detection: carry out fluorescent mark, pcr amplification and genetic analyzer according to embodiment 2 ~ 5 and detect, select the test kit that specific oligonucleotide amplimer of the present invention is right, its somatotype figure is shown in Fig. 4, select PowerPlex 18D to increase this sample simultaneously simultaneously, somatotype figure is shown in Fig. 5, and genotyping result sees the following form 4:
Table 4 the present invention and PowerPlex 18D detect this male sex's sample results
| Locus | Kit results of the present invention | PowerPlex 18D result |
| D3S1358 | 15/18 | 15/18 |
| TH01 | 9 | 9 |
| D21S11 | 29/31 | 29/31 |
| D18S51 | 15/16 | 15/16 |
| Penta E | 11/22 | 11/22 |
| Y indel | 1 | - |
| DYS391 | 10 | - |
| D12S391 | 18/19 | - |
| D6S1043 | 12/14 | - |
| D2S1338 | 23/24 | 23/24 |
| D1S1656 | 14/16 | - |
| D5S818 | 10 | 10 |
| D13S317 | 9/11 | 9/11 |
| D7S820 | 10/13 | 10/13 |
| D19S433 | 13/15.2 | 13/15.2 |
| CSF1PO | 10/11 | 10/11 |
| PentaD | 9/13 | 9/13 |
| D2S441 | 12/13 | - |
| vWA | 18/19 | 18/19 |
| D8S1179 | 12/15 | 12/15 |
| TPOX | 11 | 11 |
| FGA | 23/24 | 23/24 |
| Amelogenin | X | X |
| D16S539 | 12 | 12 |
| D22S1045 | 11/16 | - |
| SE33 | 20/30.2 | - |
| D10S1248 | 14/15 | - |
Note :-indicate without this locus
4. conclusion:
Detected sample owner has the normal sign of the male sex, is defined as male sex's sample, and after this laboratory selects PowerPlex 18D test kit to detect, result display gender-specific genes only has X, is women's sample.Although only have X with test kit detected result display Amel locus of the present invention, Y-indel and DYS391 two locus all there is specific object band, therefore can judge that this sample is as male sex's sample.
Amel locus, in Y chromosome galianconism place, more easily cause disappearance, and Y-indel and DYS391 is positioned at the long-armed place of Y chromosome, the sex determination's risk under sex site deletion can be reduced greatly.If with legal medical expert's parting detecting reagent common on the market at present, only have Amel locus, once run into this locus Y chromosome disappearance, with regard to easy, this sample is judged into women's sample.If sex sample unknown in case, direction and the progress of case will be affected completely, run into if build in storehouse at batch, find that sex is not inconsistent when checking gender, which kind of situation no matter also cause great puzzlement to a large amount of database works, be, great manpower and materials and waste of time are caused in capital, and use test kit of the present invention, the sample sex in such situation can be judged accurately, use manpower and material resources sparingly greatly and the time.
Above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.Those skilled in the art are in essential scope of the present invention, and the change made, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.
Claims (6)
1. have and strengthen the euchromosome STR locus fluorescence labeling composite amplification test kit of distinguishing ability, it is characterized in that, described test kit increases 27 str locus seats simultaneously; Comprise following 24 euchromosome STR locus: D3S1358, TH01, D21S11, D18S51, Penta E, D12S391, D6S1043, D2S1338, D1S1656, D2S441, D5S818, D13S317, D7S820, D19S433, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA, D16S539, D22S1045, SE33 and D10S1248,2 Y chromosome locus Y-indel, DYS391 and sex decision bit point Amel.
2. a kind of as claimed in claim 1 have the euchromosome STR locus fluorescence labeling composite amplification test kit strengthening distinguishing ability, and it is characterized in that, the corresponding relation of the corresponding amplimer of described 27 str locus seats is:
3. a kind of euchromosome STR locus fluorescence labeling composite amplification test kit with enhancing distinguishing ability according to claim 2, it is characterized in that: described each str locus seat adopts the pair of primers amplification being positioned at these both sides, locus core iteron, wherein there is 5 ' end fluorochrome label of a primer in often pair of primer, wherein blue fluorescent dyes FAM, Green fluorescent dye HEX, Yellow fluorochrome TAM, red fluorescence dyestuff ROX, purple fuel Alex 594, interior mark selects fluorescent orange Atto 633.
4. a kind of euchromosome STR locus fluorescence labeling composite amplification test kit with enhancing distinguishing ability according to claim 2, it is characterized in that, the method of described fluorochrome label is: first group of FAM mark: D3S1358, TH01, D21S11, D18S51 and Penta E, second group of HEX mark: Y-indel, DYS391, D12S391, D6S1043, D2S1338 and D1S1656, 3rd group of TAM mark: D5S818, D13S317, D7S820, D19S433, CSF1PO and Penta D, 4th group of ROX mark: D2S441, vWA, D8S1179, TPOX and FGA, 5th group of Alex 594 marks: Amel, D16S539, D22S1045, SE33 and D10S1248, interior mark selects fluorescent orange to mark, fluorescent marker is Atto 633.
5. a kind of euchromosome STR locus fluorescence labeling composite amplification test kit with enhancing distinguishing ability according to claim 1-4 any one, it is characterized in that, described test kit comprises following component: seedless sour water, PCR Master Mix, Primer Mix and human genome positive control;
Wherein said nuclease free water refers to the ultrapure water without RNA enzyme and DNA enzymatic;
Described PCR Master Mix includes DMSO 10mM, Tris-buffer 125mM, Repone K 125mM, ammonium sulfate 65mM, deoxynucleotide phosphates 7.5mM, BSA2.5mg/ml;
Described Primer Mix comprises all primers, Taq enzyme 2-4U/6.25 μ l, the magnesium chloride 7.5Mm of amplification 27 locus;
Shown in described Primer Mix, each primer concentration sees the following form:
6. a kind of application of euchromosome STR locus fluorescence labeling composite amplification test kit in sex identification, paternity test or forensic identification with enhancing distinguishing ability according to claim 1-5 any one.
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| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150930 |