CN110628920A - Fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof - Google Patents
Fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof, wherein the kit comprises specific primers for amplifying 35Y-STR loci, wherein the specific primers comprise 28 low mutation Y-STR loci and 7 rapid mutation Y-STR loci. The kit comprises 28Y-STR loci with low mutation rate, 7 rapid mutations and high polymorphism, and considers male family investigation and distinction between different male individuals in the same paternal line; the primers in the kit have the advantages of strong specificity, high sensitivity and accurate typing result, and can completely meet the requirements of actual case inspection, DNA database construction and paternity test; the kit has strong material detection adaptability.
Description
Technical Field
The invention belongs to the field of forensic genetics, and relates to a fluorescence labeling multiplex amplification detection system, in particular to a fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof.
Background
In the last 90 s of the century, Short Tandem Repeat (STR) loci, which are fluorescently labeled, were first proposed and used as genetic markers for the research and identification of forensic physical evidence-related aspects. Compared with other genetic markers, the STR marker has small STR locus fragments, is easy to amplify, is more suitable for trace and degraded test materials, can simultaneously carry out composite amplification on a plurality of STR loci, and can successfully carry out DNA detection and typing on mixed test materials, test materials degraded by DNA molecules, trace biological test materials and other difficult biological test materials, thereby having the advantages of rapidness, high efficiency, accuracy, sensitivity, large information content and the like. In recent years, STR typing technology is widely applied to individual identification and paternity test in forensic physical evidence, and particularly, compared with conventional RFLP and other technologies, STR multiplex amplification technology has great advantages in the aspect of DNA database construction.
The Y chromosome STR genetic marker refers to a short tandem repeat sequence existing in a non-recombination region of a human Y chromosome. Compared with the autosomal STR genetic marker, the Y chromosome STR genetic marker has three characteristics of male specificity, paternal inheritance, haplotype inheritance and the like, combines the uniqueness with the superiority of STR locus typing detection, and can be used for forensic individual identification, paternity test, DNA pedigree construction and the like.
The Y-STR has the unique advantage that all male samples in the same father line are consistent in typing and can be used as a tool for family investigation, so that the investigation range is effectively reduced, the low-mutation locus is more suitable for family investigation, but the low-mutation locus is usually low in polymorphism and is not beneficial to distinguishing families. Loci with mutation rates above 1% are known forensically as Rapidly Mutating loci (rapid mutation Y-STRs). Rapidly mutated loci have a higher capacity to discriminate between related males and generally have a higher polymorphism. Therefore, the Y-STR kit needs a large number of loci and high polymorphism of the loci so as to ensure that the kit has high enough overall polymorphism.
Disclosure of Invention
In order to overcome the defects of the prior art and obtain a kit which has high resolution and can meet the requirements of Y-STR database construction and male individual family investigation, the invention provides a fluorescence labeling composite amplification kit for 35 STR loci of human Y chromosome and application thereof.
The technical scheme is that the fluorescence labeling multiplex amplification kit comprises 35 STR loci of human Y chromosome, wherein the 35 STR loci comprise 28 low mutation Y-STR loci DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS596, DYS391, DYS456, DYS19, DYS448, DYS385a, DYS385b, DYS549, DYS437, DYS481, DYS533, DYS390, DYS458, DYS460, DYS393, Y _ GATA _ H4, DYS439, DYS635, DYS444, DYS643, DYS557, and 7 rapid mutation Y-STR loci DYS627, DYS576, DYS570, DYF387 1S1a, DYF 1b, DYS 387 518, DYS 518;
the kit comprises 31 pairs of specific primers which are used for amplifying the 35 STR loci, and the sequences of the specific primers are as follows: DYS392, SEQ ID NO 1-2; DYS389I, DYS389II and SEQ ID NO: 3-4; DYS447, SEQID NO 5-6; DYS438, SEQ ID NO: 7-8; DYS527a, DYS527b and SEQ ID NO of 9-10; DYS596 and SEQID NO: 11-12; DYS391 and SEQ ID NO 13-14; DYS456 and SEQ ID NO of 15-16; DYS19, SEQ ID NO: 17-18; DYS448 and SEQ ID NO of 19-20; DYS385a, DYS385b and SEQ ID NO 21-22; DYS549 and SEQID NO: 23-24; DYS437 and SEQ ID NO of 25-26; DYS481, SEQ ID NO 27-28; DYS533 and SEQ ID NO. 29-30; DYS390 and SEQ ID NO 31-32; DYS627 and SEQ ID NO: 33-34; DYS458, SEQ ID NO 35-36; DYS460 and SEQ ID NO: 37-38; DYS393 and SEQ ID NO of 39-40; y _ GATA _ H4, SEQ ID NO 41-42; DYS439, SEQ ID NO 43-44; DYS635 and SEQ ID NO of 45-46; DYS444, SEQ ID NO 47-48; DYS643, SEQ ID NO: 49-50; DYS557, SEQ ID NO: 51-52; DYS576, SEQ ID NO: 53-54; DYS570, SEQ ID NO: 55-56; DYF387S1a, DYF387S1b and SEQ ID NO 57-58; 59-60 of DYS449 and SEQ ID NO; DYS518 and SEQ ID NO of 61-62;
the specific amplification primers are divided into five groups: DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b and DYS596 are a first group, DYS391, DYS456, DYS19, DYS448, DYS385a, DYS385b and DYS549 are a second group, DYS437, DYS481, DYS533, DYS390, DYS627, DYS458 and DYS460 are a third group, DYS393, Y _ GATA _ H4, DYS439, DYS635, DYS444, DYS643 and DYS557 are a fourth group, DYS576, DYS570, DYS 387S1a, DYS S1b, DYS449 and DYS518 are a fifth group; at least one of the primers in each pair is labeled at its 5' end with a fluorescent dye.
Preferably, the final concentration of the specific primer in the amplification system is: DYS392, 0.072. mu.M; DYS389I and DYS389II, 0.078. mu.M; DYS447, 0.096. mu.M; DYS438, 0.06. mu.M; DYS527a and DYS527b, 0.06. mu.M; DYS596, 0.12 μ M; DYS391, 0.06. mu.M; DYS456, 0.054. mu.M; DYS19, 0.144. mu.M; DYS448, 0.054. mu.M; DYS385a and DYS385b, 0.084. mu.M; DYS549, 0.36. mu.M; DYS437, 0.072 μ M; DYS481, 0.084. mu.M; DYS533, 0.12. mu.M; DYS390, 0.12. mu.M; DYS627, 0.264 μ M; DYS458, 0.36. mu.M; DYS460, 0.42. mu.M; DYS393, 0.09. mu.M; y _ GATA _ H4, 0.144 μ M; DYS439, 0.084. mu.M; DYS635, 0.12. mu.M; DYS444, 0.18. mu.M; DYS643, 0.42. mu.M; DYS557, 0.48. mu.M; DYS576, 0.108. mu.M; DYS570, 0.156. mu.M; DYF387S1a and DYF387S1b, 0.192 μ M; DYS449, 0.3. mu.M; DYS518, 0.528. mu.M.
Primers corresponding to 35 loci and concentrations thereof included in the kit are specifically shown in table 1:
TABLE 1.35 primer information for Y STR loci
Preferably, the fluorescent dye is: any one of 6-FAM, HEX, TAMRA, ROX and ATTO, wherein each group adopts different fluorescent labels, and the internal standard adopts orange fluorescence SIZ.
Preferably, the kit comprises a Reaction mixture (Reaction Mix), a specific composite amplification primer, a hot start Taq enzyme, a DNA standard, sdH2O, alleles of 35 loci and a fluorescent molecular weight internal standard; wherein the reaction mixture comprises the following components: MgCl27.5mM, Tris-HCl 125mM, KCl 125mM, dNTPs 7.5mM, BSA 2 g/L. The components and the contents thereof are as follows: reaction Mix 10.0. mu.L, genomic DNA X. mu.l content of 0.125-5ng, primer Mix 5.0. mu.L, hot start Taq enzyme (5U/. mu.L) 0.5. mu.L, sdH2Make up to 25.0. mu.L of O.
The fluorescence labeling composite amplification kit for the 35 STR loci of the human Y chromosome is applied to forensic identification, paternity identification or DNA pedigree construction. The method comprises the following steps: collecting genome DNA, carrying out PCR amplification, and analyzing an amplification product; wherein the PCR amplification program comprises the following steps: denaturation at 95 deg.C for 2min, circulation at 94 deg.C for 30s, 60 deg.C for 1min, 65 deg.C for 1min, 30 cycles, termination of extension at 72 deg.C for 20min, and maintenance at 4 deg.C.
Preferably, the sample source used in forensic identification, paternity identification or DNA pedigree construction comprises human genomic DNA extracted using the Chelex method, magnetic bead extraction or organic extraction method; or human blood or oral cells collected by any carrier of non-extraction filter paper, FTA card, cotton swab and gauze. The source of the material to be tested comprises human blood, blood mark, semen, saliva, body fluid, hair, muscle or tissue organ.
The use method of the fluorescence labeling composite amplification kit for the 35 STR loci of the human Y chromosome is characterized in that: the method comprises the following steps:
A. preparing an amplification system, wherein the amplification system is as follows: reaction Mix 10.0. mu.L, genomic DNA X. mu.l content of 0.125-5ng, primer Mix 5.0. mu.L, hot start Taq enzyme (5U/. mu.L) 0.5. mu.L, sdH2O is complemented to 25.0 mu L;
B. performing amplification thermal cycle, wherein the amplification procedure is initial denaturation and 2 minutes at 95 ℃; thermal cycling at 94 ℃ for 30 seconds, 60 ℃ for 1min, 68 ℃ for 1min, and 30 cycles; final extension, 20min at 72 ℃; preserving heat, and maintaining at 4 ℃; wherein, the amplified sample should be preserved in dark place;
C. fluorescence detection of the amplification product on a genetic analyzer, mixing 12.5 mul of sample mixture with 1 mul of amplification product or 35Y locus Allelic typing standards Allelic Ladder (Demei union Biotechnology Co., Ltd. in Wuxi) in the system, denaturing at 95 ℃ for 3 minutes, carrying out ice bath for 3 minutes, and carrying out electrophoresis detection by using the genetic analyzer, wherein the electrophoresis adopts multi-channel or single-channel capillary electrophoresis;
the loading mixture was formulated as follows: 0.5. mu.l of molecular weight internal standard AGCU Marker SIZ-500 × (number of samples) + (12. mu.l of deionized formamide) × (number of samples);
D. typing analysis, the data collected by the genetic analyzer in step C was analyzed by the fragment analysis software GeneMapper ID-X.
The fluorescence labeling composite amplification kit for 35 STR loci of human Y chromosome has the following beneficial effects: (1) the kit comprises 28Y-STR loci with low mutation rate, 7 rapid mutations and high polymorphism, considers the male family investigation and the distinction between different male individuals in the same father line, and is a Y-STR detection product with the largest number of loci on the market; (2) the primers in the kit have the advantages of strong specificity, high sensitivity and accurate typing result, and can completely meet the requirements of actual case inspection, DNA database construction and paternity test; (3) the kit has strong material detection adaptability.
Drawings
FIG. 1 is a schematic diagram of the arrangement of 35Y loci of the kit.
FIG. 2 is a typing map of standard DNA 9948 of the kit.
FIG. 3 is a 35Y locus allelic typing standard: allelic Ladder diagram.
FIG. 4 is a Y-STR typing diagram of pedigree sample 1.
FIG. 5 is a Y-STR typing map of pedigree sample 2.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
First, locus screening
In-depth study of the locus DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y _ GATA _ H4, DYS481, DYS533, DYS576, DYS643, DYS460, DYS549, DYF387S1a, DYF387S1b, DYS449, DYS518, DYS627, DYS570, DYS527a/b, DYS447, DYS444, DYS557, DYS596, DYS446, DYS510, DYS622, DYS443, DYS587, DYS 58522, Y _ GATA _ A10, DYS526, DYS3, DYS531, DYS520, DYS617, DYS59, DYS508, DYS645, DYS508, DYS52, DYS508, DYS52, DYS 2, DYS52, DYS 2, DYS52, DYS 2: DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS596, DYS391, DYS456, DYS19, DYS448, DYS385a, DYS385b, DYS549, DYS437, DYS481, DYS533, DYS390, DYS627, DYS458, DYS460, DYS393, Y _ GATA _ H4, DYS439, DYS635, DYS444, DYS557, DYS576, DYS570, DYS 387S1a, dysf S1b, DYS449, DYS 518. The 35Y chromosome short tandem repeat sequence loci have lower genetic polymorphism and good allele frequency distribution in the population, and the requirements of the invention on loci are compounded. The corresponding information for each locus is shown in table 2:
TABLE 2.35Y STR Locus information
Second, arrangement of the loci
According to the above 35 gene loci, a unique gene locus arrangement mode and a chemical fluorescent dye labeling method are designed: DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b and DYS596 are a first group, and the fluorescent dye marker is 6-FAM; DYS391, DYS456, DYS19, DYS448, DYS385a/b and DYS549 are used as a second group, and the fluorescent dye marker is HEX; DYS437, DYS481, DYS533, DYS390, DYS627, DYS458, and DYS460 are the third group, and the fluorescent dye marker is TAMRA; DYS393, Y _ GATA _ H4, DYS439, DYS635, DYS444, DYS643 and DYS557 are the fourth group, and the fluorescent dye marker is ROX; DYS576, DYS570, DYF387S1a, DYF387S1b, DYS449, DYS518 are the fifth group, the fluorescent dye marker is ATTO, and the locus arrangement is shown in FIG. 1.
Third, design of specific primer and establishment of composite amplification condition
Downloading a locus sequence by using a UCSC or NCBI website through a locus name or a Y chromosome position; next, primer design was performed based on the sequences flanking each locus repeat unit.
(1) Specific primer design
When designing the primer, the optimal collocation of the used design software is that Premier and Oligo are combined for use, the Premier is used for carrying out automatic search, the Oligo carries out analysis and evaluation, the distribution of the base of the primer needs to be random, the Tm value is similar, the GC content is between 40 and 60 percent, and no complementary sequence exists between the primer and the primer; meanwhile, the specificity of Primer amplification is ensured, and the specificity of the 3 'end of the Primer is fully considered when Primer-BLAST software is used for comparing and analyzing the designed Primer in NCBI database, because the 3' end of the Primer has higher sequence homology and is easy to cause error triggering.
With the increase of the number of primers in the multiplex amplification system, the mutual interference between the primers of different loci is more and more serious, and the dynamics of the reaction system becomes more and more complex, so that a large number of primer sequences need to be designed for carrying out complex tests, and the amplification specificity and efficiency of the kit are finally ensured.
(2) Establishment of multiplex amplification conditions
Firstly, single amplification conditions of 35 loci are optimized, on the basis of successfully establishing single locus amplification conditions, 35 locus composite amplification PCR reaction conditions are researched, various parameters in composite amplification, such as circulating parameters, annealing temperature, buffer solution ionic strength, enzyme quantity, change of composite amplification reaction volume, template DNA quantity and the like, are determined through a large number of repeated experiments, amplification products reach the requirements of balance and specificity, a composite amplification system is established, 35 loci are amplified simultaneously, and finally primer sequences and concentrations are shown in table 1.
Fourth, adjusting the PCR reaction mixture
In the PCR System, Mg2+The concentration of the enzyme was measured by 5 gradients of 1.0mM, 1.5mM, 2.0mM, 2.5mM and 3.0mM, the concentration of dNTPs was measured by 5 gradients of 0.15mM, 0.2mM, 0.25mM, 0.3mM and 0.35mM, the content of hot start Taq enzyme was measured by 5 gradients of 1.0U, 1.5U, 2.0U, 2.5U and 3.0U, the concentration of Tris-HCl was 10mM and the concentration of KCl was 40 mM. By designing orthogonal experiments, Mg is finally synthesized2+The concentration was 2.0mM, dNTPs concentration was 0.25mM, hot start Taq enzyme content was 2.0U, Tris-HCl concentration was 10mM, KCl concentration was 40mM, and the above materials were used to prepare a Reaction mixture Reaction Mix which was added to the PCR system. The final PCR system consisted of: reaction Mix 10.0. mu.L, genomic DNA X. mu.l content 0.125-5ng, primer mixture 50 μ L, hot start Taq enzyme (5U/. mu.L) 0.5 μ L, sdH2Make up to 25.0. mu.L of O.
Example 2
The use method of the fluorescence labeling composite amplification kit for 35 STR loci of human Y chromosome comprises the following steps,
A. preparing an amplification system, wherein the amplification system is as follows: reaction Mix 10.0. mu.L, genomic DNA X. mu.l content of 0.125-5ng, primer Mix 5.0. mu.L, hot start Taq enzyme (5U/. mu.L) 0.5. mu.L, sdH2O is complemented to 25.0 mu L;
B. performing amplification thermal cycle, wherein the amplification procedure is initial denaturation and 2 minutes at 95 ℃; thermal cycling at 94 ℃ for 30 seconds, 60 ℃ for 1min, 68 ℃ for 1min, and 30 cycles; final extension, 20min at 72 ℃; preserving heat, and maintaining at 4 ℃; wherein, the amplified sample should be preserved in dark place;
C. fluorescence detection of the amplification product on a genetic analyzer, mixing 12.5 mul of sample mixture with 1 mul of amplification product or 35Y locus Allelic typing standards Allelic Ladder (Demei union Biotechnology Co., Ltd. in Wuxi) in the system, denaturing at 95 ℃ for 3 minutes, carrying out ice bath for 3 minutes, and carrying out electrophoresis detection by using the genetic analyzer, wherein the electrophoresis adopts multi-channel or single-channel capillary electrophoresis;
the loading mixture was formulated as follows: 0.5. mu.l of molecular weight internal standard AGCU Marker SIZ-500 × (number of samples) + (12. mu.l of deionized formamide) × (number of samples);
D. and (3) typing analysis, wherein the data collected by the genetic analyzer in the step C are analyzed by using a fragment analysis software GeneMapper ID-X, wherein the typing results of the DNA standard control 9948 and the allele typing standard are shown in figures 2 and 3.
Example 3 application of Individual investigation in the same family
(1) Collecting family samples in family investigation: 2 in total, samples were provided by a police office.
(2) Extracting sample DNA: adopts a silicon bead method in the forensic science DNA laboratory inspection standard (GA-T383-2002).
(3) The kit and the AGCU Database Y30 kit are used for respectively carrying out typing detection on the two family samples, and then typing results of the two kits are compared. The Y-STR typing diagrams of the two pedigree samples detected by the kit are shown in FIGS. 4 and 5.
The typing results are compared as follows:
the results showed that the two pedigree samples were tested using AGCU Database Y30, and the obtained classifications were completely consistent, and thus individual discrimination was not possible. And 7 high mutation loci are added into the kit, so that the individual identification capability of the kit is greatly improved, the same 2 family samples are amplified, and 3 loci, namely DYS627, DYF381S1a/b and DYS518, find the condition of inconsistent typing, so that individuals can be effectively distinguished, and the individual identification capability of the kit is improved.
Sequence listing
<110> material evidence identification center of province public security hall of Anhui province
Anhui-Ke bioengineering (group) corporation
Wuxi Zhongde Mei Union Biotech Ltd
<120> fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof
<141> 2019-10-31
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<210> 35
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
agagacaatg tttcacca 18
<210> 36
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ggcgtggtgg caggaac 17
<210> 37
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gatagacaga tatatctaat aggtaga 27
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gcaggaagat taaaggttgt cta 23
<210> 39
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tgtcattcct aatgtggtct tctac 25
<210> 40
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tgtctcatag aaaagacata cat 23
<210> 41
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cacttgtatc ctaggaatca tcatta 26
<210> 42
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cacctatcta tgtatctatc tatctat 27
<210> 43
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gttagaccta aaaccataac aact 24
<210> 44
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gtggattatg cctgtaatcc caac 24
<210> 45
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gtgagacact cttgatatct atggat 26
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
aacaatgtat gtgcaagtgt ctt 23
<210> 47
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gttgatgtga ttataagtga tttatg 26
<210> 48
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tgcctttgga tcccttagac agcc 24
<210> 49
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gtgcagcccc actttctggt tcac 24
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gccacccttc ccttgagttc 20
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ggaagtcttg actctcagac 20
<210> 52
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
aatgatatgt cacactgccc cgtt 24
<210> 53
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
agagatgagg tcttgctatg ttgctt 26
<210> 54
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
aaaagccaag acaaatacgc tta 23
<210> 55
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
tcattcattt tacgtttcat aac 23
<210> 56
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gtttcttgac tatgctgaat aattttca 28
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
ctgtcaccca ggctggagtg c 21
<210> 58
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
cacacttctc acactgtggc acc 23
<210> 59
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
aatcacacaa gttaaggga 19
<210> 60
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
cctcagcctc ctgagtagct ggga 24
<210> 61
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
cctgcctcag cctcatgagt agttg 25
<210> 62
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
actaagtcca ctattcttgg agc 23
Claims (7)
1. A fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome is characterized in that,
the 35 STR loci include 28 low mutation Y-STR loci DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS596, DYS391, DYS456, DYS19, DYS448, DYS385a, DYS385b, DYS549, DYS437, DYS481, DYS533, DYS390, DYS458, DYS460, DYS393, Y _ GATA _ H4, DYS439, DYS635, DYS444, DYS643, DYS557, and 7 rapid mutation Y-STR loci DYS627, DYS576, DYS570, DYF387S1a, dysf 387S1b, DYS449, DYS 518;
the kit comprises 31 pairs of specific primers which are used for amplifying the 35 STR loci, and the sequences of the specific primers are as follows: DYS392, SEQ ID NO 1-2; DYS389I, DYS389II and SEQ ID NO: 3-4; DYS447, SEQ ID NO. 5-6; DYS438, SEQ ID NO: 7-8; DYS527a, DYS527b and SEQ ID NO of 9-10; DYS596 and SEQ ID NO. 11-12; DYS391 and SEQ ID NO 13-14; DYS456 and SEQ ID NO of 15-16; DYS19, SEQ ID NO: 17-18; DYS448 and SEQ ID NO of 19-20; DYS385a, DYS385b and SEQ ID NO 21-22; DYS549 and SEQ ID NO. 23-24; DYS437 and SEQ ID NO of 25-26; DYS481, SEQ ID NO 27-28; DYS533, SEQ ID NO 29-30; DYS390 and SEQ ID NO 31-32; DYS627 and SEQ ID NO: 33-34; DYS458, SEQ ID NO 35-36; DYS460 and SEQ ID NO: 37-38; DYS393 and SEQ ID NO of 39-40; y _ GATA _ H4, SEQ ID NO 41-42; DYS439, SEQ ID NO 43-44; DYS635 and SEQ ID NO of 45-46; DYS444, SEQ ID NO 47-48; DYS643, SEQ ID NO: 49-50; DYS557, SEQ ID NO: 51-52; DYS576, SEQ ID NO: 53-54; DYS570, SEQ ID NO: 55-56; DYF387S1a, DYF387S1b and SEQ ID NO 57-58; 59-60 of DYS449 and SEQ ID NO; DYS518 and SEQ ID NO of 61-62;
the specific amplification primers are divided into five groups: DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b and DYS596 are a first group, DYS391, DYS456, DYS19, DYS448, DYS385a, DYS385b and DYS549 are a second group, DYS437, DYS481, DYS533, DYS390, DYS627, DYS458 and DYS460 are a third group, DYS393, Y _ GATA _ H4, DYS439, DYS635, DYS444, DYS643 and DYS557 are a fourth group, DYS576, DYS570, DYS 387S1a, DYS S1b, DYS449 and DYS518 are a fifth group; at least one of the primers in each pair is labeled at its 5' end with a fluorescent dye.
2. The fluorescence labeling multiplex amplification kit for 35 STR loci on human Y chromosome of claim 1, wherein the final concentration of the specific primers in the amplification system is: DYS392, 0.072. mu.M; DYS389I and DYS389II, 0.078. mu.M; DYS447, 0.096. mu.M; DYS438, 0.06. mu.M; DYS527a and DYS527b, 0.06. mu.M; DYS596, 0.12 μ M; DYS391, 0.06. mu.M; DYS456, 0.054. mu.M; DYS19, 0.144. mu.M; DYS448, 0.054. mu.M; DYS385a and DYS385b, 0.084. mu.M; DYS549, 0.36. mu.M; DYS437, 0.072 μ M; DYS481, 0.084. mu.M; DYS533, 0.12. mu.M; DYS390, 0.12. mu.M; DYS627, 0.264 μ M; DYS458, 0.36. mu.M; DYS460, 0.42. mu.M; DYS393, 0.09. mu.M; y _ GATA _ H4, 0.144 μ M; DYS439, 0.084. mu.M; DYS635, 0.12. mu.M; DYS444, 0.18. mu.M; DYS643, 0.42. mu.M; DYS557, 0.48. mu.M; DYS576, 0.108. mu.M; DYS570, 0.156. mu.M; DYF387S1a and DYF387S1b, 0.192 μ M; DYS449, 0.3. mu.M; DYS518, 0.528. mu.M.
3. The fluorescence-labeled multiplex amplification kit for 35 STR loci of human Y chromosome of claim 1, wherein said fluorescent dye is: any one of 6-FAM, HEX, TAMRA, ROX and ATTO, and the fluorescent label adopted by each group is different.
4. The fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome of claim 1, wherein the kit comprises reaction mixture, 35 loci alleles, hot start Taq enzyme, DNA standard, sdH2O and a fluorescent molecular weight internal standard;
the composition of the reaction mixture was: MgCl2 7.5mM,Tris-HCl 125mM，KCl 125mM，dNTPs 7.5mM，BSA 2g/L。
5. The use method of the fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome as claimed in any one of claims 1 to 4, which comprises the following steps,
A. preparing an amplification system, wherein the amplification system is as follows: reaction Mix 10.0. mu.L, genomic DNA X. mu.l content of 0.125-5ng, primer Mix 5.0. mu.L, hot start Taq enzyme (5U/. mu.L) 0.5. mu.L, sdH2O is complemented to 25.0 mu L;
B. performing amplification thermal cycle, wherein the amplification procedure is initial denaturation and 2 minutes at 95 ℃; thermal cycling at 94 ℃ for 30 seconds, 60 ℃ for 1min, 68 ℃ for 1min, and 30 cycles; final extension, 20min at 72 ℃; preserving heat, and maintaining at 4 ℃; wherein, the amplified sample should be preserved in dark place;
C. fluorescence detection of the amplification product on a genetic analyzer, mixing 12.5 mul of sample mixture with 1 mul of amplification product or 35Y locus allele typing standard Allelic Ladder in the system, denaturing at 95 ℃ for 3 minutes, carrying out ice bath for 3 minutes, and carrying out electrophoresis detection by adopting the genetic analyzer;
the loading mixture was formulated as follows: 0.5. mu.l of molecular weight internal standard AGCU Marker SIZ-500 × (number of samples) + (12. mu.l of deionized formamide) × (number of samples);
D. typing analysis, the data collected by the genetic analyzer in step C was analyzed by the fragment analysis software GeneMapper ID-X.
6. The use of the fluorescence-labeled multiplex amplification kit for 35 STR loci of human Y chromosome of claim 1 in forensic identification, paternity testing or DNA pedigree construction.
7. The use of claim 6, wherein the sample source used in forensic identification, paternity testing or DNA pedigree construction comprises human genomic DNA extracted using the Chelex method, magnetic bead extraction or organic extraction method; or human blood or oral cells collected by any carrier of non-extraction filter paper, FTA card, cotton swab and gauze.
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| CN112852971A (en) * | 2020-12-31 | 2021-05-28 | 百特元生物科技（北京）有限公司 | Primer group and kit for simultaneously amplifying 44 human Y-STR loci and application of primer group and kit |
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