CN110564861B - Fluorescent marker composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof - Google Patents
Fluorescent marker composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection, and particularly discloses a fluorescent marker composite amplification kit for human Y chromosome STR loci and InDel loci, which comprises 41 pairs of specific primers, and can amplify 37Y chromosome STR loci and 8Y chromosome InDel loci in a single tube. The invention has the characteristics of strong specificity, accurate typing, high sensitivity and the like, and meanwhile, 8 InDel sites are added to distinguish the families of the sample to a certain extent. The invention can meet the needs of cases detection, database construction, paternity test and the like.
Description
Technical Field
The invention belongs to the field of forensic genetics, and relates to a fluorescent marker multiplex amplification detection system, in particular to a fluorescent marker multiplex amplification kit for human Y chromosome STR loci and InDel loci and application thereof.
Background
The short tandem repeat locus (short tandem repeat, STR) has been widely used for forensic study and identification as a second generation genetic marker following a restriction fragment length polymorphism since the eighth nineteenth of the twentieth century, and is one of the most widely used genetic markers at present. Compared with other genetic markers, the STR marker has the advantages of small STR locus fragment, easy amplification, more suitability for trace and degradation detection materials, capability of carrying out composite amplification on a plurality of STR loci simultaneously, rapidness, high efficiency, accuracy, sensitivity, large information quantity and the like, and is widely applied to individual identification and parent identification in forensic material evidence. Y chromosome STR genetic markers refer to short tandem repeats present in non-recombinant regions of the human Y chromosome. Compared with the autosomal STR genetic marker, the Y chromosome STR genetic marker has three characteristics of male specificity, male parent genetics, haplotype genetics and the like, and can be used for forensic individual identification, paternity test, DNA pedigree construction and the like.
The InDel polymorphism is a special type of genetic marker for the alleles in the human genome, represented by small fragments of different sizes inserted or deleted in the genome. InDel is used as a new-generation forensic genetic identification marker, and compared with STR, the advantages of InDel are mainly represented in the following steps: (1) Relatively low mutation rate (2.3X10) -9 ) Ratio STR (10 -3 ) And SNPs (2.3X10) -8 ) Is more stable; (2) The amplified target fragment is relatively short, and is more suitable for the detection and analysis of degradation detection materials; (3) Because of the regional difference of gene frequencies, the Y-InDel genetic marker not only can be used for identifying individualsThe method can also be used for analyzing ancestor related information sites to distinguish crowds; (4) Easy to type, can be used for conventional electrophoresis typing or high-throughput automatic typing method, and has no stutter product.
Y-STR and Y-InDel possess respective characteristics and advantages. The Y-STR is significantly higher than Y-InDel with respect to single locus polymorphisms. In the case of genetic stability, the mutation rate of Y-InDel is much lower than that of Y-STR. According to the characteristics of the two, the two are combined, and when the kit contains enough Y-STR loci and has higher individual recognition capability, part of Y-InDel loci are added, so that the kit can be used for rapidly distinguishing whether part of samples come from the same family or not and has a certain effect on family investigation.
Currently, some of the commercial kits already contain both Y-STR and Y-InDel, such as: AGCU Y37+5, Y Filer Platinum, DNA Typer 36Y, goldeney DNA 38Y and the like, but the kit generally contains 3-5Y-InDel loci, the number is small, and the sample families are difficult to effectively distinguish, 8Y-InDel loci are added on the basis of using 37Y-STRs, so that the distinguishing capability of the kit on the families is greatly improved, and meanwhile, the 37Y-STRs have stronger recognition capability on different male individuals.
Disclosure of Invention
The technical problems to be solved are as follows: in order to overcome the defects of the prior art, the invention provides a fluorescent marker composite amplification kit for human Y chromosome STR loci and InDel loci and application thereof, and the kit has high resolution and can meet the requirements of Y chromosome DNA database construction and male individual family investigation.
The technical scheme is as follows: a fluorescent-labeled multiplex amplification kit for human Y chromosome STR loci and InDel sites, the kit comprising specific primers for amplifying 37Y-STR loci and 8Y-InDel sites;
the 37Y chromosome STR loci are DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS522, DYS596, DYS391, DYS456, DYS19, DYS448, DYS520, DYS627, DYS557, DYS437, DYS481, DYS533, DYS390, DYS385a, DYS385b, DYF387S1a, DYF387S1b, DYS460, DYS393, Y_GATA_H2, DYS439, DYS635, DYS444, DYS643, DYS549, DYS576, DYS458, DYS570, DYS449, DYS518,
the 8Y chromosome InDel sites are: rs20063M, rs75055M, rs21464M, rs771783753, rs20151M, rs759551978, rs199815934, rs74557M.
The kit comprises 41 pairs of specific primers which are used for amplifying the 37 STR loci and the 8 InDel loci, wherein the sequences of the specific primers are as follows: rs20063M, SEQ ID NO 1-2; DYS392, SEQ ID NO 3-4; DYS389I and DYS389II, SEQ ID NO 5-6; DYS447, SEQ ID NO 7-8; DYS438, SEQ ID NO 9-10; DYS527a and DYS527b, SEQ ID NO 11-12; DYS522, SEQ ID NO 13-14; 15 to 16 of DYS596 and SEQ ID NO; rs75055M, SEQ ID NO 17-18; DYS391, SEQ ID NO 19-20; DYS456, SEQ ID NO 21-22; DYS19, SEQ ID NO 23-24; DYS448, SEQ ID NO 25-26; DYS520, SEQ ID NO 27-28; DYS627, SEQ ID NO 29-30; DYS557, SEQ ID NO 31-32; rs21464M, SEQ ID NO 33-34; DYS437, SEQ ID NO 35-36; DYS481, SEQ ID NO 37-38; DYS533, SEQ ID NO 39-40; DYS390, SEQ ID NO 41-42; DYS385a and DYS385b, SEQ ID NO 43-44; DYF387S1a, DYF387S1b and SEQ ID NO 45-46; DYS460, SEQ ID NO 47-48; rs771783753, SEQ ID NO 49-50; rs20151M, SEQ ID NO 51-52; DYS393, SEQ ID NO 53-54; Y_GATA_H24, SEQ ID NO 55-56; DYS439, SEQ ID NO 57-58; DYS635, SEQ ID NO 59-60; DYS444 and SEQ ID NO 61-62; DYS643, SEQ ID NO 63-64; DYS549, SEQ ID NO 65-66; rs759551978, SEQ ID NO 67-68; rs199815934, SEQ ID NO 69-70; rs74557M, SEQ ID NO 71-72; DYS576, SEQ ID NO 73-74; DYS458, 75-76 SEQ ID NO; DYS570, SEQ ID NO 77-78; DYS449, SEQ ID NO 79-80; DYS518, SEQ ID NO 81-82.
Preferably, the final concentration of the specific primer in the amplification system is: rs20063M, 0.06 μM; DYS392, 0.06. Mu.M; DYS389I and DYS389II, 0.078. Mu.M; DYS447, 0.18. Mu.M; DYS438, 0.06. Mu.M; DYS527a and DYS527b, 0.06 μm; DYS522, 0.09. Mu.M; DYS596, 0.21. Mu.M; rs75055M, 0.06. Mu.M; DYS391, 0.066. Mu.M; DYS456, 0.054. Mu.M; DYS19, 0.204. Mu.M; DYS448, 0.072. Mu.M; DYS520, 0.072. Mu.M; DYS627, 0.21. Mu.M; DYS557, 0.3. Mu.M; rs21464M, 0.036 μΜ; DYS437, 0.066. Mu.M; DYS481, 0.078. Mu.M; DYS533, 0.18. Mu.M; DYS390, 0.132. Mu.M; DYS385a and DYS385b, 0.12. Mu.M; DYF387S1a and DYF387S1b, 0.15. Mu.M; DYS460, 0.192. Mu.M; rs771783753, 0.132 μm; rs20151M, 0.09 μΜ; DYS393, 0.09. Mu.M; y_gata_h4, 0.12 μΜ; DYS439, 0.084. Mu.M; DYS635, 0.096. Mu.M; DYS444, 0.12. Mu.M; DYS643, 0.18. Mu.M; DYS549, 0.3. Mu.M; rs759551978, 0.12 μΜ; rs199815934, 0.06 μΜ; rs74557M, 0.06. Mu.M; DYS576, 0.12. Mu.M; DYS458, 0.144. Mu.M; DYS570, 0.192. Mu.M; DYS449, 0.24. Mu.M; DYS518, 0.30. Mu.M.
The primer sequences and the concentrations of the primer sequences corresponding to the 45 loci included in the kit are shown in Table 1.
Table 1.37 primer information for Y STR loci and 8 InDel loci.
Preferably, the specific composite amplification primers are divided into five groups: rs20063M, DYS, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS522 and DYS596 are the first group, rs75055M, DYS391, DYS456, DYS19, DYS448, DYS520, DYS627 and DYS557 are the second group, rs21464M, DYS437, DYS481, DYS533, DYS390, DYS385a, DYS385b, DYF387S1a, DYF387S1b and DYS460 are the third group, rs771783753, D rs20151M, YS393, y_gata_h4, DYS439, DYS635, DYS444, DYS643 and DYS549 are the fourth group, rs759551978, rs199815934, rs74557, DYS576, DYS458, DYS570, DYS449 and DYS518 are the fifth group; at least one of the primers in each pair is labeled with a fluorescent dye at its 5' end.
Preferably, the fluorescent dye is: 6-FAM, HEX, SUM, LYN, PUR, and the fluorescent label used is different for each group.
Preferably, the kit comprises a Reaction mixture (Reaction Mix), a specific composite amplification primer, a hot start Taq enzyme, a DNA standard substance and sdH 2 Alleles of the 37 STR loci plus 8 InDel loci on the O, human Y chromosome and a fluorescent molecular weight internal standard;
the composition of the Reaction mixture (Reaction Mix) was: mgCl 2 7.5mM,Tris-HCl 125mM，KCl 125mM，dNTPs 7.5mM，BSA 2g/L。
Preferably, the fluorescent molecular weight internal standard is orange fluorescent SiZ.
The fluorescent labeling composite amplification kit for the human Y chromosome STR locus and the InDel locus comprises the following specific use steps:
A. preparing an amplification system, wherein the PCR amplification system comprises 10.0 mu L of Reaction Mix, 0.125-5ng of genome DNA X mu L, 5.0 mu L of primer mixture, 0.5 mu L of hot start Taq enzyme (5U/. Mu.L) and 25.0 mu L of sdH 2O;
B. the amplification thermal cycle, the amplification program of the thermal cycle instrument is initial denaturation, 95 ℃ for 2 minutes; thermal cycling, 94℃for 30 seconds, 60℃for 1min,68℃for 1min,30 cycles; final extension, 20 min at 72 ℃; preserving heat and maintaining at 4 ℃;
C. fluorescence detection of the amplified product on a genetic analyzer, mixing 12.5 μl of the sample mixture with 1 μl of the amplified product or 45Y locus allele typing standard substances Alllic Ladder in the system, denaturing at 95deg.C for 3 min, ice-bath for 3 min, performing electrophoresis detection with the genetic analyzer, and performing electrophoresis with multiple or single capillary electrophoresis;
the loading mixture was formulated as follows: 0.5 μl molecular weight internal standard AGCU Marker SIZ-500× (number of samples) + (12 μl deionized formamide) × (number of samples);
D. typing analysis, analysis of the collected data using the fragment analysis software GeneMapper ID-X analysis of the genetic Analyzer in step C.
The invention also provides application of the fluorescent marker composite amplification kit for the human Y chromosome STR locus and the InDel locus in forensic identification, paternity test or DNA pedigree construction.
Preferably, the sample sources used in forensic identification, paternity identification, or DNA pedigree construction include human genomic DNA extracted using Chelex, magnetic bead extraction, or organic extraction; or human blood or oral cells collected by any one carrier of filter paper, FTA card, cotton swab and gauze without extraction. Sources of test materials include human blood, blood marks, semen, saliva, body fluids, hair, muscle, or tissue organs.
The beneficial effects are that: (1) The kit comprises 37Y chromosome STR loci and 8Y chromosome InDel loci, and both the male family investigation and the distinction between different male individuals of the same male parent are considered; (2) The primer in the kit has 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 adaptability of detecting materials.
Drawings
FIG. 1 is a schematic diagram of a locus arrangement.
FIG. 2 is a typing chart of standard DNA9948 of the kit.
FIG. 3 is an allelic typing standard for 37Y-STRs with 8Y-InDel: alllic Ladder diagram.
FIG. 4 is an amplification typing of sample 1 in a home screening test.
FIG. 5 is an amplification typing of sample 2 in a home screening test.
Detailed Description
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Example 1
1. Y-STR locus selection
By combining DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y_GATA_H2, DYS481, DYS533, DYS576, DYS643, DYS460, DYS549, DYF387S1a, DYF387S1b, DYS449, DYS518, DYS627, DYS570, DYS527a/b, DYS447, DYS444, DYS557 the 59Y chromosome short tandem repeat loci and the allele genetic characteristics thereof of DYS596, DYS446, DYS510, DYS622, DYS443, DYS587, DYS522, Y_GATA_A10, DYS520, DYS552, DYS593, DYS531, DYS459a/b, DYS508, DYS388, DYS617, DYS645, DYS713, DYS630, DYF404S1, DYS626, and DYS526a/b are intensively studied, 37Y chromosome short tandem repeat loci were screened: the 37Y chromosome short tandem repeat loci DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS522, DYS596, DYS391, DYS456, DYS19, DYS448, DYS520, DYS627, DYS557, DYS437, DYS481, DYS533, DYS390, DYS385a, DYS385b, DYF387S1a, DYF387S1b, DYS460, DYS393, Y_GATA_H2, DYS439, DYS635, DYS444, DYS643, DYS549, DYS576, DYS458, DYS570, DYS449, DYS518 have higher genetic polymorphisms and good allele frequency distribution in the population, in accordance with the requirements of the present invention for loci. The corresponding information for each locus is shown in Table 2:
table 2.37Y-STR locus information.
2. Screening of Y-InDel sites
Using genetic data from 1000genome Project, cluster, YSNP evolutionary tree and other databases, MAF in the east Asian population: between 0.2 and 0.5, and removing sites with repeated polymorphism, removing sites without reference number, and the like, and screening Y-InDel loci which can be used for individual identification. Screening can be used to aid in inferring the ethnicity-derived Y-InDel locus to which a male sample belongs, on the principle that allele frequency differs by greater than 0.3 between east Asia and European, east Asian and African, european and African populations. Finally, 8Y-InDel loci were screened out.
Table 3.8 allele frequencies for Y-InDel loci in different populations.
3. Locus arrangement
Based on the above 45 loci, a unique locus arrangement mode and a method for labeling by using a chemical fluorescent dye are designed: rs20063M, DYS, DYS389I/II, DYS447, DYS438, DYS527a/b, DYS522 and DYS596 are the first group, and the fluorescent dye marker is 6-FAM; rs75055M, DYS, DYS456, DYS19, DYS448, DYS520, DYS627, DYS557 are the second group, and the fluorescent dye marker is HEX; rs21464M, DYS437, DYS481, DYS533, DYS390, DYS385a/b, DYF387S1a, DYF387S1b, DYS460 as third group, fluorescent dye marker as SUM; rs771783753, rs20151M, DYS393, Y_GATA_H2 4, DYS439, DYS635, DYS444, DYS643, DYS549 are the fourth group, and the fluorescent dye marker is LYN; rs759551978, rs199815934, rs74557M, DYS576, DYS458, DYS570, DYS449, DYS518 are fifth group, fluorescent dye marker is PUR, and the locus is arranged as shown in fig. 1.
4. Specific primer design and establishment of complex amplification conditions
Downloading a locus sequence by using UCSC or NCBI websites through locus names or Y chromosome positions; next, primer design was performed based on sequences flanking each locus repeat unit.
(1) Specific primer design
When designing the primer, the optimal collocation of the used design software is that the Premier and the Oligo are used together, the Premier is used for automatic searching, the Oligo is used for analysis and evaluation, the base distribution of the primer is random, the Tm values are similar, the GC content is between 40% and 60%, and the primer have no complementary sequence; meanwhile, the specificity of Primer amplification is guaranteed, and the designed Primer is compared and analyzed by using Primer-BLAST software in NCBI database, so that the specificity of the 3 'end of the Primer is fully considered, and error priming is easily caused if the sequence homology of the 3' end of the Primer is high.
Along with the increase of the number of primers in a multiplex amplification system, the mutual interference among the primers of different loci is more and more serious, and the dynamics of a reaction system is more and more complex, so that a large number of primer sequences are required to be designed for complex tests, and finally the amplification specificity and efficiency of the kit are ensured.
(2) Establishment of Complex amplification conditions
The single amplification conditions of 45 loci are optimized, the amplification PCR reaction conditions of the 45 loci are studied on the basis of successfully establishing the single locus amplification conditions, various parameters in the multiplex amplification, such as circulation parameters, annealing temperature, buffer ionic strength, enzyme quantity, change of the multiplex amplification reaction volume, template DNA quantity and the like, are determined through a large number of repeated experiments, so that the amplified products reach the balanced and specific requirements, a multiplex amplification system is established, 45 loci are amplified simultaneously, and the final primer sequences and concentrations are shown in Table 1.
5. Adjusting the PCR reaction mixture
In the PCR system, mg 2+ The concentrations were measured for 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM 5 gradients, dNTPs were measured for 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM 5 gradients, hot start Taq enzyme levels were measured for 1.0U, 1.5U, 2.0U, 2.5U, 3.0U total 5 gradients, tris-HCl concentration was 10mM, KCl concentration was 40mM, respectively. By designing orthogonal experiments, finally Mg 2+ The concentration was set at 2.0mM, dNTPs at 0.25mM, the hot start Taq enzyme content at 2.0U, tris-HCl at 10mM, KCl atThe Reaction mixture was prepared at 40mM using the above materials, and added to the PCR system. The final PCR system consisted of: reaction mix 10.0. Mu.L, genomic DNA with a content of 0.125-5ng, primer mixture 5.0. Mu.L, hot start Taq enzyme (5U/. Mu.L) 0.5. Mu.L, sdH 2 O was made up to 25.0. Mu.L.
Example 2Y-InDel application in family investigation
(1) 2 blood spot samples from different families: the sample is provided by a public security bureau, which are all Chinese citizens and have no other national blood system.
(2) The above 2 samples were subjected to typing detection by using the kit, and Y-InDel and Y-STR of the 2 samples were compared, and the results are shown in Table 4.
The specific using steps of the fluorescent labeling composite amplification kit are as follows:
A. preparing an amplification system, wherein the PCR amplification system comprises Reaction Mix10.0 mu L, genome DNA X mu L with a content of 0.125-5ng (the optimal template amount range is 0.125-5ng, when the sample DNA content is in the range, high-quality amplification patterns can be obtained), primer mixture 5.0 mu L, hot start Taq enzyme (5U/. Mu.L) 0.5 mu L, sdH 2 O was made up to 25.0. Mu.L.
B. The amplification thermal cycle, the amplification program of the thermal cycle instrument is initial denaturation, 95 ℃ for 2 minutes; thermal cycling, 94℃for 30 seconds, 60℃for 1min,68℃for 1min,30 cycles; final extension, 20 min at 72 ℃; preserving heat and maintaining at 4 ℃.
C. Fluorescence detection of amplified products on a genetic analyzer, which consisted of deionized formamide with the in-system molecular weight internal standard AGCU Marker SIZ-500, [ 0.5. Mu. l AGCU Marker SIZ-500 (De Mei Biotechnology Co., ltd.) X (number of samples) + (12. Mu.l deionized formamide) × (number of samples) ], and mixing 12.5. Mu.l of the sample mixture with 1. Mu.l of amplified products or 45Y locus allele genotyping standard allelicladder (DeMei Biotechnology Co., ltd.) in the system, avoiding the generation of bubbles, denaturing at 95℃for 3 minutes in an ice bath for 3 minutes, and allowing the genetic analyzer to perform electrophoresis detection as soon as possible.
D. Typing analysis, analyzing the collected data by using a genetic analyzer in the step C by using fragment analysis software GeneMapper ID-X, and performing electrophoresis by using a multi-channel or single-channel capillary electrophoresis.
Table 4 shows the typing detection results.
| Y-InDel name | Parting 1 | Parting 2 | Y-STR name | Parting 1 | Parting 2 |
| rs20063M | 2 | 1 | DYS392 | 13 | 12 |
| rs75055M | 2 | 2 | DYS389I | 13 | 12 |
| rs21464M | 2 | 2 | DYS447 | 24 | 24 |
| rs771783753 | 2 | 2 | DYS389II | 30 | 30 |
| rs20151M | 2 | 1 | |
10 | 10 |
| rs759551978 | 2 | 2 | DYS527a/b | 22/22 | 20/23 |
| rs199815934 | 1 | 1 | DYS522 | 12 | 12 |
| rs74557M | 2 | 2 | DYS596 | 15 | 14 |
| DYS391 | 11 | 10 | |||
| DYS456 | 15 | 15 | |||
| DYS19 | 15 | 15 | |||
| DYS448 | 19 | 19 | |||
| DYS520 | 22 | 22 | |||
| DYS627 | 17 | 20 | |||
| DYS557 | 14 | 15 | |||
| DYS437 | 14 | 15 | |||
| DYS481 | 22 | 24 | |||
| DYS533 | 12 | 11 | |||
| DYS390 | 22 | 23 | |||
| DYS385a/b | 12/19 | 12/18 | |||
| DYF387S1 | 36/39 | 35/38 | |||
| |
10 | 9 | |||
| DYS393 | 12 | 12 | |||
| Y_GATA_H4 | 12 | 12 | |||
| DYS439 | 10 | 14 | |||
| DYS635 | 21 | 19 | |||
| DYS444 | 14 | 13 | |||
| DYS643 | 11 | 11 | |||
| DYS549 | 13 | 14 | |||
| DYS576 | 20 | 18 | |||
| DYS458 | 17 | 17 | |||
| DYS570 | 16 | 17 | |||
| DYS449 | 31 | 32 | |||
| DYS518 | 35 | 36 |
The results show that the two samples are detected by using the kit, the Y-InDel genotypes of the two samples are different, the two samples can be rapidly judged to be not from the same family according to the results, the judgment can be effectively proved by the differences of the genotypes of other Y-STR loci in the kit, and the application can greatly improve the efficiency of family investigation.
Sequence listing
<110> institute of public security science and technology in Hunan province
Criminal investigation support team for public security bureau in long-sand city
Tin-free middle De Mey Biotechnology Co., ltd
<120> fluorescent labeling composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof
<141> 2019-08-09
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aactgatgca agaaagattc actga 25
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gtacaaaaca ggttaataat tcct 24
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
taaggcagga gaatagctgg aattc 25
<210> 12
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
agattagcca caacataagt aaggtagt 28
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gaaatctgtg catggggaag 20
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gcccagtcag ccctgctac 19
<210> 15
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
taaattcaca tcataattat ctttc 25
<210> 16
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gatttttgtt tgttttctag tgag 24
<210> 17
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
cactttgttg tgtggaaaaa agaca 25
<210> 18
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
gcatcagtct ttctcctcag aaata 25
<210> 19
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
ccatccttat ctcttgtgta tctattc 27
<210> 20
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
aggcaggcag ataggcaga 19
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
aactcggact ggctcatctt gc 22
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
agaaaacccc atcaactcag cc 22
<210> 23
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
tgatttcact atgactactg agtttctg 28
<210> 24
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
ccatggccat gtagtgagga caag 24
<210> 25
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
cgcgagacag aaagggagat agaga 25
<210> 26
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ggagaccttt tcttccttaa cgtg 24
<210> 27
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
caccaagccc gctatagaca gtga 24
<210> 28
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
ctcccatgta gctgagatta cag 23
<210> 29
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
cacagataat gccactgcac tccac 25
<210> 30
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gagtccactg gagaccttaa tattat 26
<210> 31
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
attgattgtt aatctgattt ctgaacc 27
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
cagctaggtg ctggatccaa t 21
<210> 33
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
ttatcaaatt cacaactccc cagtat 26
<210> 34
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
aagactaagg gaatgagcat atgaaag 27
<210> 35
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
catgcccatc cggtctatct a 21
<210> 36
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gaacagagga agaccctgtc attcac 26
<210> 37
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ctttaagagg agtctgctaa a 21
<210> 38
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
acacagacag ctcaccagaa gg 22
<210> 39
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
cattctaatt atgtctcttc taacta 26
<210> 40
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
atggctgtaa gtagagatca ccaa 24
<210> 41
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
atccatggat attaggttta ttacatt 27
<210> 42
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
atggtagcat aatagaaatt ttatga 26
<210> 43
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gaaaggaagg aaggaaggag 20
<210> 44
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
gctaggtaaa gctggtaagg gc 22
<210> 45
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gtaagtgcat tttatcccat ttactc 26
<210> 46
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
gagtctgtaa gtggctcaga c 21
<210> 47
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
tgcctatcat ttattatgta tttgtc 26
<210> 48
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
ctcagcttta atccaactag aa 22
<210> 49
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
aaagatcata ttactagtga ctgttctca 29
<210> 50
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
aggcttgtca ctaatttttg ttga 24
<210> 51
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
tgtcaccatc tcacctgtat caaa 24
<210> 52
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
gaaagctcag tgttgaagtc ctg 23
<210> 53
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
tcttctactt gtgtcaatac ag 22
<210> 54
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
ataatttaaa aaaataaaac tcaagtcca 29
<210> 55
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
aatgttatgc tgaggagaat ttcc 24
<210> 56
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
acaggataaa tcacctatct atg 23
<210> 57
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
gaattaatag attcaaggtg atagat 26
<210> 58
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
gctagtctca aactcctgac ctca 24
<210> 59
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
ctgaatggga gcagaaatgc ccaatg 26
<210> 60
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
caaggctcca tctcaaacaa ca 22
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
ctccacttta accagtatac 20
<210> 62
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
gcagagaaaa tacatttact attc 24
<210> 63
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
caagaagtca ccatccgtg 19
<210> 64
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
gcaggatggt gagggataaa a 21
<210> 65
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
gtgtgcatag aggtgttcag 20
<210> 66
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
cgagatgtca ctaccaccc 19
<210> 67
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
ctcaccaaag gaatgcacat c 21
<210> 68
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
agataattaa aattgacagt tatcagtttg 30
<210> 69
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
caactcaact ccagtgattt aaactc 26
<210> 70
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
gctgatacct ttgtttctgt tcattctt 28
<210> 71
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
tctttgtggt acttgttttg tgtga 25
<210> 72
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
aaacagtatg ccaaaaacaa cagaa 25
<210> 73
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
tgggctgagg agttcaatct c 21
<210> 74
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
atttgtcttg gctttttctt 20
<210> 75
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
gtcaagattg agccattgca ctg 23
<210> 76
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
gaccttgtga tccagccacc 20
<210> 77
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
cgttaccatt aaacttcagt tatgtg 26
<210> 78
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
ccagacaact ggtggcaacc t 21
<210> 79
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
tcaagcctgt tctatgaata ttttc 25
<210> 80
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
agcctggaag tggagtttgc tgt 23
<210> 81
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
gcaggtgaat cacttgaacc tg 22
<210> 82
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
tcaggtctgt cactttgtca cttt 24
Claims (9)
1. A fluorescent labeling composite amplification kit for human Y chromosome STR loci and InDel loci is characterized in that 37Y chromosome STR loci are DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS522, DYS596, DYS391, DYS456, DYS19, DYS448, DYS520, DYS627, DYS557, DYS437, DYS481, DYS533, DYS390, DYS385a, DYS385b, DYF387S1a, DYF387S1b, DYS460, DYS393, Y_GATA_H2, DYS439, DYS635, DYS444, DYS643, DYS549, DYS576, DYS458, DYS570, DYS449, DYS518,
the 8Y chromosome InDel sites are: rs20063M, rs75055M, rs21464M, rs771783753, rs20151M, rs759551978, rs199815934, rs74557M;
the kit comprises 41 pairs of specific primers which are used for amplifying the 37 STR loci and the 8 InDel loci, and the sequence of the specific primers of rs20063M is SEQ ID NO. 1-2; the sequence of the specific primer of DYS392 is SEQ ID NO 3-4; the sequence of the specific primers of DYS389I and DYS389II is SEQ ID NO 5-6; the sequence of the specific primer of DYS447 is SEQ ID NO 7-8; the sequence of the specific primer of DYS438 is SEQ ID NO 9-10; the specific primer sequences of DYS527a and DYS527b are SEQ ID NO. 11-12; the sequence of the specific primer of DYS522 is SEQ ID NO. 13-14; the sequence of the specific primer of DYS596 is SEQ ID NO. 15-16; the sequence of the specific primer of rs75055M is SEQ ID NO. 17-18; the sequence of the specific primer of DYS391 is SEQ ID NO. 19-20; the sequence of the specific primer of DYS456 is SEQ ID NO. 21-22; the sequence of the specific primer of DYS19 is SEQ ID NO. 23-24; the sequence of the specific primer of DYS448 is SEQ ID NO 25-26; the sequence of the specific primer of DYS520 is SEQ ID NO 27-28; the sequence of the specific primer of DYS627 is SEQ ID NO 29-30; the sequence of the specific primer of DYS557 is SEQ ID NO. 31-32; the sequence of the specific primer of rs21464M is SEQ ID NO. 33-34; the sequence of the specific primer of DYS437 is SEQ ID NO. 35-36; the sequence of the specific primer of DYS481 is SEQ ID NO. 37-38; the sequence of the specific primer of DYS533 is SEQ ID NO 39-40; the sequence of the specific primer of DYS390 is SEQ ID NO. 41-42; the specific primer sequences of the DYS385a and the DYS385b are SEQ ID NO. 43-44; the sequence of the specific primers of DYF387S1a and DYF387S1b is SEQ ID NO 45-46; the sequence of the specific primer of DYS460 is SEQ ID NO. 47-48; the sequence of the specific primer of rs771783753 is SEQ ID NO. 49-50; the sequence of the specific primer of rs20151M is SEQ ID NO. 51-52; the sequence of the specific primer of the DYS393 is SEQ ID NO. 53-54; the sequence of the specific primer of the Y_GATA_H24 is SEQ ID NO. 55-56; the sequence of the specific primer of DYS439 is SEQ ID NO 57-58; the sequence of the specific primer of DYS635 is SEQ ID NO 59-60; the sequence of the specific primer of DYS444 is SEQ ID NO 61-62; the sequence of the specific primer of DYS643 is SEQ ID NO. 63-64; the sequence of the specific primer of DYS549 is SEQ ID NO 65-66; the sequence of the specific primer of rs759551978 is SEQ ID NO 67-68; the sequence of the specific primer of rs199815934 is SEQ ID NO 69-70; the sequence of the specific primer of rs74557M is SEQ ID NO. 71-72; the sequence of the specific primer of DYS576 is SEQ ID NO 73-74; the sequence of the specific primer of DYS458 is 75-76 of SEQ ID NO; the sequence of the specific primer of DYS570 is SEQ ID NO. 77-78; the sequence of the specific primer of DYS449 is SEQ ID NO. 79-80; the sequence of the specific primer of DYS518 is SEQ ID NO. 81-82.
2. The fluorescent-labeled multiplex amplification kit for human Y chromosome STR locus and InDel locus according to claim 1, characterized in that the final concentration of specific primers for rs20063M in the amplification system is 0.06 μΜ; the final concentration of the specific primer of DYS392 in the amplification system is 0.06 mu M; the final concentration of specific primers for DYS389I and DYS389II in the amplification system was 0.078. Mu.M; the final concentration of the specific primer of DYS447 in the amplification system is 0.18. Mu.M; the final concentration of the specific primer of DYS438 in the amplification system was 0.06. Mu.M; the final concentration of specific primers for DYS527a and DYS527b in the amplification system was 0.06. Mu.M; the final concentration of the specific primer of DYS522 in the amplification system is 0.09 mu M; the final concentration of the specific primer of DYS596 in the amplification system is 0.21 mu M; the final concentration of the specific primer of rs75055M in the amplification system is 0.06 mu M; the final concentration of the specific primer of DYS391 in the amplification system is 0.066 mu M; the final concentration of the specific primer of DYS456 in the amplification system is 0.054. Mu.M; the final concentration of the specific primer of DYS19 in the amplification system is 0.204 mu M; the final concentration of the specific primer of DYS448 in the amplification system is 0.072 mu M; the final concentration of the specific primer of DYS520 in the amplification system is 0.072 mu M; the final concentration of the specific primer of DYS627 in the amplification system is 0.21 mu M; the final concentration of the specific primer of DYS557 in the amplification system is 0.3 mu M; the final concentration of the specific primer of rs21464M in the amplification system is 0.036 mu M; the final concentration of the specific primer of DYS437 in the amplification system is 0.066 mu M; the final concentration of the specific primer of DYS481 in the amplification system is 0.078. Mu.M; the final concentration of the specific primer of DYS533 in the amplification system is 0.18. Mu.M; the final concentration of the specific primer of DYS390 in the amplification system is 0.132 mu M; the final concentration of specific primers for DYS385a and DYS385b in the amplification system was 0.12. Mu.M; the final concentration of specific primers for DYF387S1a and DYF387S1b in the amplification system was 0.15. Mu.M; the final concentration of the specific primer of DYS460 in the amplification system is 0.192 mu M; the final concentration of the specific primer of rs771783753 in the amplification system is 0.132 mu M; the final concentration of the specific primer of rs20151M in the amplification system is 0.09 mu M; the final concentration of the specific primer of DYS393 in the amplification system was 0.09. Mu.M; the final concentration of the specific primer of Y_GATA_H24 in the amplification system is 0.12 mu M; the final concentration of the specific primer of DYS439 in the amplification system was 0.084. Mu.M; the final concentration of the specific primer of DYS635 in the amplification system is 0.096. Mu.M; the final concentration of the specific primer of DYS444 in the amplification system is 0.12 mu M; the final concentration of the specific primer of DYS643 in the amplification system was 0.18. Mu.M; the final concentration of the specific primer of DYS549 in the amplification system is 0.3 mu M; the final concentration of the specific primer of rs759551978 in the amplification system is 0.12 mu M; the final concentration of the specific primer of rs199815934 in the amplification system is 0.06 mu M; the final concentration of the specific primer of rs74557M in an amplification system is 0.06 mu M; the final concentration of the specific primer of DYS576 in the amplification system was 0.12. Mu.M; the final concentration of the specific primer of DYS458 in the amplification system is 0.144. Mu.M; the final concentration of the specific primer of DYS570 in the amplification system is 0.192 mu M; the final concentration of the specific primer of DYS449 in the amplification system was 0.24. Mu.M; the final concentration of the specific primer for DYS518 in the amplification system was 0.30. Mu.M.
3. The fluorescent-labeled multiplex amplification kit for human Y chromosome STR locus and InDel locus according to claim 2, wherein the specific multiplex amplification primers are divided into five groups: specific primers for rs20063M, DYS392, DYS389I, DYS447, DYS389II, DYS438, DYS527a, DYS527b, DYS522 and DYS596 are the first group, specific primers for rs75055M, DYS391, DYS456, DYS19, DYS448, DYS520, DYS627 and DYS557 are the second group, specific primers for rs21464M, DYS437, DYS481, DYS533, DYS390, DYS385a, DYS385b, DYF387S1a, DYF387S1b and DYS460 are the third group, specific primers for rs771783753, rs20151M, DYS393, y_gata_h4, DYS439, DYS635, DYS444, DYS643 and DYS549 are the fourth group, and specific primers for rs759551978, 199815934, rs74557M, DYS576, DYS458, DYS570, DYS449 and DYS518 are the fifth group. At least one of the primers in each pair is labeled with a fluorescent dye at its 5' end.
4. The fluorescent-labeled multiplex amplification kit of human Y chromosome STR locus and InDel locus according to claim 3, wherein the fluorescent dye is: 6-FAM, HEX, SUM, LYN, PUR, and the fluorescent label used is different for each group.
5. The fluorescent-labeled multiplex amplification kit for human Y chromosome STR loci and InDel sites according to claim 1, wherein the kit comprises specific multiplex amplification primers, reaction mixture, alleles of human Y chromosome 37 STR loci plus 8 InDel sites, hot start Taq enzyme, DNA standard, sdH 2 O and fluorescent molecular weight internal standard;
the composition of the reaction mixture is: mgCl 2 7.5mM,Tris-HCl 125mM，KCl 125mM，dNTPs 7.5mM，BSA 2g/L。
6. The fluorescent-labeled multiplex amplification kit for human Y chromosome STR locus and InDel locus as defined in claim 5, wherein the fluorescent molecular weight internal standard is orange fluorescent SIZ.
7. The fluorescent-labeled multiplex amplification kit for human Y chromosome STR loci and InDel sites according to any one of claims 1 to 6, comprising the following specific steps:
A. preparing an amplification system, wherein the PCR amplification system comprises 10.0 mu L of Reaction Mix, the template amount of genome DNA is in the range of 0.125-5ng, 5.0 mu L of primer mixture, 5U/mu L of hot start Taq enzyme, 0.5 mu L of hot start Taq enzyme and sdH 2 O is added to 25.0 mu L;
B. the amplification thermal cycle, the amplification program of the thermal cycle instrument is initial denaturation, 95 ℃ for 2 minutes; thermal cycling, 94℃for 30 seconds, 60℃for 1min,68℃for 1min,30 cycles; final extension, 20 min at 72 ℃; preserving heat and maintaining at 4 ℃;
C. fluorescence detection of the amplified product on a genetic analyzer, mixing 12.5 μl of the sample mixture with 1 μl of the amplified product or 45Y locus allele typing standard substances Alllic Ladder in the system, denaturing at 95deg.C for 3 min, ice-bath for 3 min, performing electrophoresis detection with the genetic analyzer, and performing electrophoresis with multiple or single capillary electrophoresis;
the loading mixture was formulated as follows: 0.5 μl molecular weight internal standard AGCU Marker SIZ-500×sample number+12 μl deionized formamide×sample number;
D. typing analysis, analysis of the collected data using the fragment analysis software GeneMapper ID-X analysis of the genetic Analyzer in step C.
8. Use of the fluorescent-labeled multiplex amplification kit of human Y chromosome STR locus and InDel locus according to claim 1 in forensic identification, paternity identification or DNA pedigree construction.
9. The use according to claim 8, wherein the sample sources used in forensic identification, paternity identification or DNA pedigree construction comprise human genomic DNA extracted by Chelex, magnetic bead extraction or organic extraction; or human blood or oral cells collected by any one carrier of filter paper, FTA card, cotton swab and gauze without extraction.
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| CN112662786A (en) * | 2021-01-28 | 2021-04-16 | 苏州阅微基因技术有限公司 | Fluorescence multiplex amplification system, kit and application of 54Y chromosome loci |
| CN113025725A (en) * | 2021-03-16 | 2021-06-25 | 广州深晓基因科技有限公司 | Fluorescence detection kit based on Y-STR locus and Y-Indel, and use method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220413A (en) * | 2018-02-05 | 2018-06-29 | 浙江省公安物证鉴定中心 | The fluorescent composite amplification reagent kit of joint-detection people's Y chromosome STR and Indel locus and its application |
| CN109321662A (en) * | 2018-10-31 | 2019-02-12 | 浙江省公安物证鉴定中心 | A kind of fluorescence labeling composite amplification kit of 15 Indel locus of human Y-chromosome |
| CN109880912A (en) * | 2019-03-07 | 2019-06-14 | 基点认知技术(北京)有限公司 | The composite amplification reagent kit of 44 human Y-chromosome locus and its application |
-
2019
- 2019-08-09 CN CN201910732648.0A patent/CN110564861B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220413A (en) * | 2018-02-05 | 2018-06-29 | 浙江省公安物证鉴定中心 | The fluorescent composite amplification reagent kit of joint-detection people's Y chromosome STR and Indel locus and its application |
| CN109321662A (en) * | 2018-10-31 | 2019-02-12 | 浙江省公安物证鉴定中心 | A kind of fluorescence labeling composite amplification kit of 15 Indel locus of human Y-chromosome |
| CN109880912A (en) * | 2019-03-07 | 2019-06-14 | 基点认知技术(北京)有限公司 | The composite amplification reagent kit of 44 human Y-chromosome locus and its application |
Non-Patent Citations (1)
| Title |
|---|
| 新一代遗传标记――InDel研究进展;孙宽等;《法医学杂志》;20130425(第02期);第134-139,143页 * |
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