WO2017097135A1 - 一种人类基因组33个基因座的复合扩增试剂盒及其应用 - Google Patents

一种人类基因组33个基因座的复合扩增试剂盒及其应用 Download PDF

Info

Publication number
WO2017097135A1
WO2017097135A1 PCT/CN2016/107578 CN2016107578W WO2017097135A1 WO 2017097135 A1 WO2017097135 A1 WO 2017097135A1 CN 2016107578 W CN2016107578 W CN 2016107578W WO 2017097135 A1 WO2017097135 A1 WO 2017097135A1
Authority
WO
WIPO (PCT)
Prior art keywords
loci
human genome
amplification kit
composite amplification
kit
Prior art date
Application number
PCT/CN2016/107578
Other languages
English (en)
French (fr)
Inventor
杜蔚安
刘超
李发院
刘宏
张勇果
钱水
王邦超
郑卫国
卢青
忻星
Original Assignee
广东华美众源生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东华美众源生物科技有限公司 filed Critical 广东华美众源生物科技有限公司
Publication of WO2017097135A1 publication Critical patent/WO2017097135A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to a composite amplification kit for simultaneously analyzing human genome autosomal and Y chromosome loci, in particular to a composite amplification kit for 33 loci of human genome and application thereof.
  • the short tandem repeat is a commonly used molecular genetic marker. Its fragment is small and easy to amplify. It is suitable for the detection of trace and degraded bio-samples.
  • the amplification conditions of each locus are similar and can realize complex amplification and automated detection. Therefore, it is sensitive, accurate, fast, and has a large amount of information, which is very suitable for establishing a DNA database.
  • the human Y chromosome is a small proximal centromere chromosome composed of a long arm and a tiny short arm. In addition to the autosomal region, the Y chromosome does not undergo exchange and recombination in meiosis, and is uniploidally transmitted downward, showing the paternal genetic characteristics, and the sequence variation is completely caused by the cumulative mutation.
  • the Y chromosome STR locus (abbreviated as Y-STR) refers to a short tandem repeat sequence present in the non-recombinant region of the human Y chromosome. At present, there are more than 220 Y-STR loci confirmed and named in various forms.
  • the commonly used Y-STR loci have 9 European minimum haplotype loci, including DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393 and two US Research Groups on DNA Analysis Methods (SWGDAM) recommended loci, including DYS438, DYS439. Comparing the discovered Y-STR genetic markers with A-STR genetic markers, most Y-STR genetic markers have complex repeating structures, containing two or more different repeating units, core repeats of individuals in the population or The number of repetitions is different, forming a population genetic polymorphism. Combining the uniqueness of Y-STR genetic markers with the superiority of STR typing can be applied to individual identification, paternity testing, family planning and genealogy building.
  • STR genetic markers As with the above-mentioned characteristics of STR genetic markers and their detection applications, many research institutes and reagent companies have invested a lot of manpower and resources to research and develop kit products that can be applied to forensic evidence, such as the current Expressmarker 20 fluorescence detection reagents commonly used in the domestic market. Box, AGCU Database Y24 Fluorescence Detection Kit and PowerPlex21System (PP21) and PowerPlex Y23System (PPY23) in foreign markets, they can be divided into Do not use for individual identification detection and paternal detection. Usually when a major criminal case occurs, the forensic doctor will first perform an A-STR test on the physical evidence extracted from the site.
  • a Y-STR test is added to increase the detection clue.
  • the existing STR typing technology is affected by factors such as the state of the sample, the amplification system, and the detection flux, the two inspection procedures increase the detection time and often delay the best chance of capturing the suspect, and also because of the crime.
  • the small amount of material evidence left by the suspect on the scene leads to a decrease in detection efficiency or even failure to help provide more investigative clues.
  • the existing test kits have not been able to meet the requirements of the rapid test and on-site inspection capabilities of forensic DNA in public security combat. How to achieve A-STR locus and Y-STR locus together to shorten the detection time and improve the test efficiency Become a problem to be solved.
  • a composite amplification kit for 33 loci of human genome the kit containing 33 sets of primer pairs for specifically amplifying 33 loci, 33 loci are D3S1358, D13S317, D7S820, D16S539, Penta E, DYS635, DYS456, TPOX, TH01, D2S1338, CSF1PO, DYS385a/b, DYS458, DYS391, D19S433, vWA, D21S11, D18S51, D6S1043, Amelogenin, D8S1179, D5S818, D12S391, FGA, DYS438, DYS393, DYS389I, DYS389II , DYS439, DYS392, Y_GATA_H4 and DYS390, the primer pairs corresponding to 33 loci are shown in Table 1:
  • the 5' end of at least one primer in each locus is labeled with a fluorescent dye.
  • the primer pairs of the 33 loci are labeled with 5 different fluorescent dyes, and are divided into 5 groups according to the labeled fluorescent dyes, respectively:
  • D3S1358, D13S317, D7S820, D16S539, Penta E and DYS635 are the first group;
  • DYS456, TPOX, TH01, D2S1338, CSF1PO, DYS385a/b and DYS458 are the second group;
  • DYS391, D19S433, vWA, D21S11, D18S51, D6S1043 are the third group
  • Amelogenin, D8S1179, D5S818, D12S391, FGA, DYS438 are the fourth group;
  • DYS393, DYS389I, DYS389II, DYS439, DYS392, Y_GATA_H4 and DYS390 are the fifth group.
  • the five different fluorescent dyes are 6-FAM, HEX, TAMRA, ROX and VIG, respectively.
  • the kit also includes a reaction buffer, a hot-start Taq enzyme, ultrapure water, an Allelic Ladder, and a fluorescent molecular weight internal standard.
  • the reaction buffer is 50 mM Tris-HCl, 50 mM KCl, 2.0 mM MgCl 2 , 0.2 mM. dNTPs and 0.8 mg/mL BSA.
  • the amplification procedure of the polymerase chain reaction in the kit is: 95 ° C for 2 min; 94 ° C for 30 s, 61 ° C for 40 s, 72 ° C for 1 min, repeating 15 cycles; 90 ° C for 30 s, 60 ° C for 1 min, 65 ° C for 80 s, Repeat 15 cycles; 60 ° C for 30 min.
  • Another object of the present invention is to provide an application of the above-described composite amplification kit for 33 loci of the human genome in individual recognition, paternity testing, suspect family screening or autosomal databases and Y chromosome databases.
  • the present invention comprises 18 A-STR loci recommended by the Ministry of Public Security DNA database, and includes 14 Y-STR loci with low mutation rate and high polymorphism, and Amelogenin locus to achieve single tube simultaneous Amplification detection of 33 loci, the largest in the industry;
  • the invention realizes one experiment and simultaneously analyzes two data of A-STR and Y-STR, and can be used for rapid investigation of cases at the same time, thereby shortening the detection time and improving the efficiency of eliminating and determining suspects, 14 Y-STRs.
  • the locus has a large geographical and surname orientation for the investigation of the case;
  • the invention realizes the construction of two DNA databases of A-STR and Y-STR, which greatly saves manpower, material and financial resources;
  • the invention can detect a small amount of male DNA in the background of a large number of female DNA samples, and is a powerful weapon for detecting mixed spots such as rape crime;
  • the present invention does not need to perform Y-STR experimental analysis separately because the Y-STR data is already included;
  • the present invention relates to a method for analyzing a DNA sample using a six-color fluorescent labeling complex amplification assay system; wherein the DNA sample to which the present invention is applied is derived from spots, saliva spots, tissues, hair follicles, blood marks or blood;
  • kit primer of the present invention has strong amplification specificity and temperature tolerance through careful design and continuous modification.
  • the present invention selects 18 A-STR loci and 1 sex identification locus recommended by the Ministry of Public Security DNA database, and adds 14 Y with low mutation rate and high polymorphism.
  • the -STR locus enables simultaneous amplification and detection of both autosomes and Y chromosomes in one experiment.
  • the kit of the invention can directly amplify blood spots and saliva spots with filter paper and FTA card as a carrier without template extraction and purification process, and can also be applied to DNA samples extracted by different extraction methods, and belongs to domestic and foreign exclusive products.
  • the kit of the invention can be used for rapid investigation of cases, improving detection efficiency and speed of case investigation, especially for the male sample micro-detection and paternal-child paternity detection in the rape case, the kit has wide application range and good compatibility, and The existing forensic DNA detection system is fully compatible.
  • Figure 1 shows the results of STR typing of the reference number 9948 provided by a public security bureau
  • Figure 2 is an electropherogram of an allelic ladder
  • Figure 3 is a STR typing result for the actual sample detection
  • Figure 4 is a typing map showing non-specific amplification of the DYS458 locus when a female sample is tested;
  • Figure 5 is a typing map showing non-specific amplification of the DYS393 locus when a female sample is tested;
  • Figure 6 is a fragmentation map showing non-specific amplification of the DYS389I/II locus when a female sample is tested.
  • locus name is followed by two loci labeled a/b.
  • test kit The main components of the test kit:
  • the 33 loci were grouped and fluorescently labeled as follows: D3S1358, D13S317, D7S820, D16S539, Penta E, and DYS635 as the first group, fluorescent dye markers 6-FAM; DYS456, TPOX, TH01, D2S1338, CSF1PO , DYS385a/b and DYS458 are the second group, the fluorescent dye label is HEX; DYS391, D19S433, vWA, D21S11, D18S51, D6S1043 are the third group, the fluorescent dye label is TAMRA; Amelogenin, D8S1179, D5S818, D12S391, FGA DYS438 is the fourth group, the fluorescent dye label is ROX; DYS393, DYS389I/II, DYS439, DYS392, H4, DYS390 are the fifth group, and the fluorescent dye label is VIG.
  • composition of the PCR system is shown in Table 4:
  • the reaction buffer was 50 mM Tris-HCl, 50 mM KCl, 2.0 mM MgCl 2 , 0.2 mM dNTPs, and 0.8 mg/mL BSA. .
  • the method for detecting amplification products in the kit is determined by capillary genetic analyzer or gel electrophoresis.
  • capillary genetic analyzer or gel electrophoresis.
  • other purposes can be used for other purposes.
  • the PCR amplification product was centrifuged at 3000 rpm for 5 minutes, and 1 ⁇ L of the product or kit allele genotyping standard was mixed with 0.5 ⁇ L of the fluorescent molecular weight internal standard AGCU Marker SIZ-500 and 12 ⁇ L of deionized formamide to avoid bubble generation. Denaturation at 95 ° C for 3 minutes, ice bath for 3 minutes, genetic analysis of the analyzer, analysis of the electrophoresis data using the fragment analysis software GeneMapper.
  • the 14 Y-STR loci in the fluorescently labeled complex amplification kit of 33 loci were applied using the protocol of Example 1 for case investigation.
  • a sample of 1056 samples from different individuals was provided by a public security bureau: including 350 blood samples, 330 fine spots, 260 saliva spots, and 116 tissues.
  • Genomic DNA extraction is performed in accordance with the GA/T 383-2014 Forensic Science DNA Laboratory Test Specification.
  • PCR amplification, electrophoresis detection, and result analysis were performed as in Example 1. All samples were analyzed by simultaneous amplification amplification using the Yfiler kit, the kit of invention patent 201310364183.0 and the kit of 201410208458.6. The results are shown in Table 6:
  • the resolution performance advantage of the present invention is significant.
  • the test results of 400 samples from different sources show that the four Y-STR loci newly added by the scheme of invention patent 201310364183.0 can divide the 400 samples into 174 haplotypes, and the 10 patents added by the scheme of invention patent 201410208458.6 STR and 14 Y-STRs of the present invention can be divided into 400 haplotypes; when the sample is increased to 600, 4 Y-STRs can be divided into 241 haplotypes, and 10 Y-STRs can be divided.
  • 14 Y-STRs can still be divided into 600 haplotypes; out of a total of 1056 samples, 4 Y-STRs can be divided into 432 haplotypes, and 10 Y-STRs can 886 haplotypes were separated, and the 14 Y-STRs of the present invention were able to separate 907 haplotypes, and the resolution was Yfiler. More than 99% of the 17 Y-STRs in the kit.
  • kit of the invention in forensic individual identification:
  • the sample is a blood spot numbered 9948 provided by a public security bureau.
  • the kit realizes the simultaneous detection and analysis of the A-STR locus and the Y-STR locus.
  • the original inspection procedure needs to be reduced to one completion, which reduces the detection time and improves the detection efficiency, thereby increasing the capture of suspects.
  • Opportunity; another test reduced the absolute amount of sample required, thereby increasing the amount of valid evidence that the suspect left behind at the scene of the case, and eventually more material evidence increased the investigative clues.
  • the existing test kits can not meet the requirements of the rapid test and on-site inspection capability of forensic DNA in public security combat, and the A-STR and Y-STR loci can be tested together. Reduce inspection time and improve the efficiency of eliminating and determining suspects.
  • the primers were repeatedly optimized, the concentration was continuously adjusted, and the results of each locus were balanced on the basis of non-specific amplification. Since DYS458, DYS393, DYS389I/II have high homology with the X chromosome, the present invention finds suitable regions to design specific primers through repeated comparison of the genome database, ensuring that all Y-STR loci are still in 100 ng female samples. No amplification. The optimization process of these three loci is described as follows.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明提供了一种人类基因组33个基因座的复合扩增试剂盒，所述基因座包含18个A-STR基因座和14个Y-STR基因座以及Amelogenin基因座。所述试剂盒实现了单管同时扩增检测33个基因座，对常染色体和Y染色体在一个实验中同步扩增和检测。

Description

一种人类基因组33个基因座的复合扩增试剂盒及其应用 技术领域
本发明涉及一种同时分析人基因组常染色体及Y染色体基因座的复合扩增试剂盒，特别涉及一种人类基因组33个基因座的复合扩增试剂盒及其应用。
背景技术
短串联重复序列(STR)是目前普遍应用的分子遗传标记，其片段小易扩增，适宜于检验微量和降解生物检材，各基因座的扩增条件相似而能够实现复合扩增和自动化检测，因而具有灵敏、准确、快速、信息量大等优点，非常适用于建立DNA数据库。上世纪90年代美国FBI选择了13个常染色体STR基因座(简称A-STR)用于建立DNA数据索引***——CODIS(Combined DNA Index System)：CSF1PO、D3S1358、D5S818、D7S820、D8S1179、D13S317、D16S539、D18S51、D21S11、FGA、TH01、TPOX、vWA，目前该***已被多个国家的DNA数据库在其基础上借鉴和发展。
人类Y染色体是小的近端着丝粒染色体，它由长臂和微小的短臂两部分组成。Y染色体除拟常染色体区外，在减数***中不发生交换重组，呈单倍型独立向下传递，表现出父系遗传特征，其序列的变异完全由累积的突变所致。Y染色体STR基因座(简称Y-STR)是指存在于人类Y染色体非重组区域的短串联重复序列。目前，以各种形式确认并命名的Y-STR基因座已有220多个，常用的Y-STR基因座有9个欧洲最小单倍型基因座，包括DYS19、DYS385a/b、DYS389I/II、DYS390、DYS391、DYS392、DYS393和2个美国DNA分析方法技术工作组(Scientific Working Group on DNA Analysis Methods，简称SWGDAM)推荐的基因座，包括DYS438、DYS439。将已发现的Y-STR遗传标记与A-STR遗传标记相比，大多数Y-STR遗传标记具有复杂重复结构，含有两种或两种以上的不同重复单位，群体中个体的核心重复序列或重复次数不同，形成群体遗传多态性。将Y-STR遗传标记的独特性与STR分型检测的优越性相结合，可应用于个体识别、亲子鉴定、家系排查及家谱构建等。
正如上述STR遗传标记及其检测应用的特点，众多科研院所及试剂公司投入大量人力物力对其进行研究并开发可应用于法医物证的试剂盒产品，如目前国内市场常见的Expressmarker 20荧光检测试剂盒、AGCU Database Y24荧光检测试剂盒以及国外市场的PowerPlex21System(PP21)和PowerPlex Y23System(PPY23)，它们可以分 别用于个体识别检测和父系检测等。通常当一个重大刑事案件发生时，法医会先对现场提取的物证进行A-STR检测，当确认为男性犯罪嫌疑人遗留的物证时，再加做Y-STR检测以增加侦查线索。然而，因现有的STR分型技术受到检材状态、扩增体系、检测通量等因素的影响，两次检验程序增加了检测时间而往往延误抓获犯罪嫌疑人的最佳机会，同时由于犯罪嫌疑人遗留在现场的物证量少导致检测效能降低甚至失败而不能帮助提供更多侦查线索。现有检测试剂盒已无法很好的满足公安实战对于法医DNA的快速检验和现场检验能力的要求，如何实现A-STR基因座和Y-STR基因座一起检测，达到缩短检测时间同时提高检验效能成为有待解决的问题。
发明内容
本发明的目的在于提供一种人类基因组33个基因座的复合扩增试剂盒。
本发明所采取的技术方案是：
一种人类基因组33个基因座的复合扩增试剂盒，所述试剂盒含有用于特异性扩增33个基因座的33组引物对，33个基因座分别为D3S1358、D13S317、D7S820、D16S539、Penta E、DYS635、DYS456、TPOX、TH01、D2S1338、CSF1PO、DYS385a/b、DYS458、DYS391、D19S433、vWA、D21S11、D18S51、D6S1043、Amelogenin、D8S1179、D5S818、D12S391、FGA、DYS438、DYS393、DYS389I、DYS389II、DYS439、DYS392、Y_GATA_H4及DYS390，33个基因座对应的引物对如表1所示：
表1、各个基因座的引物序列及浓度
Figure PCTCN2016107578-appb-000001
Figure PCTCN2016107578-appb-000002
Figure PCTCN2016107578-appb-000003
每个基因座中的至少一条引物的5’端标记有荧光染料。
优选的，33个基因座的引物对使用5种不同的荧光染料标记，根据标记的荧光染料不同分为5组，分别是：
D3S1358、D13S317、D7S820、D16S539、Penta E和DYS635为第一组；
DYS456、TPOX、TH01、D2S1338、CSF1PO、DYS385a/b和DYS458为第二组；
DYS391、D19S433、vWA、D21S11、D18S51、D6S1043为第三组；
Amelogenin、D8S1179、D5S818、D12S391、FGA、DYS438为第四组；
DYS393、DYS389I、DYS389II、DYS439、DYS392、Y_GATA_H4及DYS390为第五组。
所述5种不同的荧光染料分别是6-FAM、HEX、TAMRA、ROX和VIG。
试剂盒内还包括反应缓冲液、热启动Taq酶、超纯水、Allelic Ladder以及荧光分子量内标，所述反应缓冲液为50mM的Tris-HCl、50mM的KCl、2.0mM的MgCl2、0.2mM的dNTPs和0.8mg/mL的BSA。
优选的，试剂盒内聚合酶链式反应的扩增程序为：95℃2min；94℃30s，61℃40s，72℃1min，重复15个循环；90℃30s，60℃1min，65℃80s，重复15个循环；60℃30min。
本发明的另一个目的在于提供上述人类基因组33个基因座的复合扩增试剂盒在个体识别、亲子鉴定、嫌疑人家系排查或常染色体数据库及Y染色体数据库中的的应用。
本发明的有益效果是：
1.本发明包含了公安部DNA数据库推荐使用的18个A-STR基因座，同时涵盖了14个突变率低、多态性高的Y-STR基因座，以及Amelogenin基因座，实现单管同时扩增检测33个基因座，为业内最多；
2.本发明实现了一次实验同时分析A-STR及Y-STR两种数据，可同时用于案件的快速排查，达到缩短检测时间同时提高排除和确定犯罪嫌疑人的效率，14个Y-STR基因座对于案件的排查具有很大的地域、姓氏指向性；
3.本发明实现了同建A-STR及Y-STR两个DNA数据库，大大节约了人力物力财力；
4.本发明可以检测出大量女性DNA样本背景下的微量男性DNA，是如***犯罪等混合斑检测的有力武器；
5.本发明在检测父-子亲子鉴定时，由于Y-STR数据已包含而无需单独再做Y-STR实验分析；
6.本发明涉及一种采用六色荧光标记复合扩增检验***对DNA样品进行分析的方法；其中，本发明适用的DNA样品来源于精斑、唾液斑、组织、毛囊、血痕或血液等；
7.本发明试剂盒引物通过精心设计和不断修改，具有较强的扩增特异性及温度耐受性。
综上所述，本发明选择了公安部DNA数据库推荐的18个A-STR基因座和1个性别鉴定基因座，并在此基础上添加了14个突变率低、多态性较高的Y-STR基因座，实现了一次实验同时对常染色体和Y染色体同步扩增和检测。本发明试剂盒可以对以滤纸和FTA卡为载体的血斑和唾液斑进行直接扩增而无需模板提取和纯化过程，也可以适用于不同提取方法提取的DNA样品，属于国内、外独家产品。本发明试剂盒可用于案件的快速排查，提高检测效率及案件排查速度，尤其对***案中男性样本微量检测及父-子亲子鉴定检测成效显著，本试剂盒适用范围广，兼容性好，与现有法医DNA检测***完全兼容。
附图说明
图1是某公安局提供的编号为9948的对照品的STR分型结果；
图2是等位基因分型标准物的电泳图谱；
图3是对实际样本检测STR分型结果；
图4是对女性样本进行检测时，DYS458基因座出现非特异扩增的分型图谱；
图5是对女性样本进行检测时，DYS393基因座出现非特异扩增的分型图谱；
图6是对女性样本进行检测时，DYS389I/II基因座出现非特异扩增的分型图谱。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。但本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
本发明所使用的33个基因座的相应信息如下表2所示：
表2、各基因座信息
Figure PCTCN2016107578-appb-000004
Figure PCTCN2016107578-appb-000005
上述的基因座中，基因座名称后标记有a/b的为两个基因座。
检测试剂盒的主要成分：
33个基因座对应的引物对如表3所示：
表3、各个基因座的引物序列及浓度
Figure PCTCN2016107578-appb-000006
Figure PCTCN2016107578-appb-000007
将所述33个基因座按如下分组并进行荧光标记：D3S1358、D13S317、D7S820、D16S539、Penta E和DYS635为第一组，荧光染料标记物为6-FAM；DYS456、TPOX、TH01、D2S1338、CSF1PO、DYS385a/b和DYS458为第二组，荧光染料标记物为HEX；DYS391、D19S433、vWA、D21S11、D18S51、D6S1043为第三组，荧光染料标记物为TAMRA；Amelogenin、D8S1179、D5S818、D12S391、FGA、DYS438为第四组，荧光染料标记物为ROX；DYS393、DYS389I/II、DYS439、DYS392、H4、DYS390为第五组，荧光染料标记物为VIG。
PCR体系各组成如表4所示：
表4、试剂盒包含组分
Figure PCTCN2016107578-appb-000008
所述反应缓冲液为50mM的Tris-HCl、50mM的KCl、2.0mM的MgCl2、0.2mM的dNTPs和0.8mg/mL的BSA。。
PCR扩增程序如表5所示：
表5、试剂盒扩增程序
Figure PCTCN2016107578-appb-000009
根据相关规定，用于法医鉴证时，本试剂盒检测扩增产物的方法采用毛细管遗传分析仪或凝胶电泳进行测定。当然，用于其他目的时，也可以使用其他公知的方法进行。
电泳结束后，将PCR扩增产物3000rpm离心5分钟，取1μL产物或试剂盒等位基因分型标准物与0.5μL荧光分子量内标AGCU Marker SIZ-500和12μL去离子甲酰胺混合，避免产生气泡，95℃变性3分钟，冰浴3分钟，遗传分析仪电泳检测，用片段分析软件GeneMapper分析电泳数据。
实施例2
采用实施例1中的方案应用33个基因座的荧光标记复合扩增试剂盒中的14个Y-STR基因座进行案件排查。由某公安局提供1056份来自不同个体的样品：包括350份血样，330份精斑、260份唾液斑、116份组织。基因组DNA的提取参考《GA/T 383-2014法庭科学DNA实验室检验规范》进行。按照实施例1进行PCR扩增、电泳检测和结果分析。所有样品用Yfiler试剂盒、发明专利201310364183.0的试剂盒和201410208458.6的试剂盒同步扩增分析。结果如表6所示：
表6、1056人份模板4Y、10Y、14Y分型结果中的单倍型数量的统计
随着分析数据的增加，本发明的分辨效能优势明显。不同来源的400份样品测试结果表明，发明专利201310364183.0的方案新加入的4个Y-STR基因座可以把该400份样品分出174种单倍型，发明专利201410208458.6的方案加入的10个Y-STR和本发明的14个Y-STR都可以分出400种单倍型；而当样品增加到600份时，4个Y-STR可以分出241种单倍型，10个Y-STR可以分出580种单倍型，而14个Y-STR仍可以分出600种单倍型；在总共1056份样品中，4个Y-STR可以分出432种单倍型，10个Y-STR可以分出886种单倍型，而本发明的14个Y-STR可以分出907种单倍型，分辨能力达到Yfiler试 剂盒中17个Y-STR的99％以上。
实施例3
本发明试剂盒在法医个体识别中的应用：
1、收集案件中的血斑：样品是由某公安局提供的编号为9948的血斑。
2、样品DNA提取：基因组DNA的提取参考《GA/T 383-2014法庭科学DNA实验室检验规范》进行。
3、PCR扩增及毛细管电泳分析，结果如图1、图2、图3。
本试剂盒实现了A-STR基因座和Y-STR基因座同步检测分析，原本需要两次的检验程序缩减到一次完成，减少了检测时间，提高了检测效率，从而增加了抓获犯罪嫌疑人的机会；另外一次检验减少了样品所需的绝对量，从而增加了犯罪嫌疑人遗留在案件现场的有效物证量，最终更多的物证信息增加了侦查线索。随着刑事案件的增多，现有检测试剂盒已不能很好的满足公安实战对于法医DNA的快速检验和现场检验能力的要求，而A-STR和Y-STR两种基因座一起检测，可以达到缩短检测时间的同时提高排除和确定犯罪嫌疑人的效率。
实施例4
为建立一个同步扩增A-STR和Y-STR两种基因座的复合扩增体系，反复优化引物，不断调整浓度，在不出现非特异扩增基础上保持各基因座结果均衡。由于DYS458、DYS393、DYS389I/II与X染色体同源性较高，本发明通过对基因组数据库的反复比对，找到合适区域设计特异引物，保证了所有Y-STR基因座在100ng的女性样品中仍无扩增。如下分述这三个基因座的优化过程。
采用如表6所示的引物，对采集的女性DNA样品进行扩增，扩增程序同实施例1。扩增后结果如图3所示(该图中所对应的是SEQ IDNO.71～72)。从图中可以看出，DYS385a/b基因座对应的位置出现了非特异扩增峰(图3红色箭头)，说明对照例中的引物会导致结果的误判。在采用表6中另外四对引物进行扩增时，也同样出现了非特异性扩增峰。这主要是由于DYS458与X染色体同源性较高，对女性样本进行检测时，会出现误扩增的情况。
表6、在DYS458基因座扩增中出现非特异峰的引物表
Figure PCTCN2016107578-appb-000011
同理，采用表7中DYS393的引物，也会造成女性样本的非特异扩增峰，扩增程序同实施例1。扩增结果如图4所示(该图中所对应的是SEQ ID NO.79～80)，DYS393基因座在女性样本中出来了扩增峰，证实是该基因座引物在基因组中的非特异扩增峰。在采用表7中其他四对引物进行扩增时，也同样出现了非特异性扩增峰。用DYS393的序列在NCBI数据库中BLAST发现，其与很多常染色体以及X染色体都有较高的同源性，导致该基因座较难找到合适引物设计位置。通过基因座序列比对，将引物3`末端设计在Y染色体特异的碱基位置并多次重复实验验证，最终确定本发明所选择的特异引物。
表7、在DYS393基因座扩增中出现非特异峰的引物表
Figure PCTCN2016107578-appb-000012
同理，采用表8中DYS389I/II的引物，也会造成女性样本的非特异扩增峰，扩增程序同实施例1。扩增结果如图5(DYS390位置)所示(该图中所对应的是SEQ ID NO.85～86)，DYS389I/II基因座在女性样本中出现扩增峰，证实是该基因座引物在基因组中的非特异扩增峰。在采用表8中另两对引物进行扩增时，也同样出现了非特异性扩增峰。经反复分析DYS389I/II基因座的模板序列，发现该段序列在男女性样本中差异比较小，如果引物设计的位置不对，就会导致非特异扩增问题，从而导致结果的误判。
表8、在DYS389基因座扩增中出现非特异峰的引物表
Figure PCTCN2016107578-appb-000013
以上对本发明的较佳实施方式进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明精神的前提下还可作出种种的等同变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (9)

  1. 一种人类基因组33个基因座的复合扩增试剂盒，其特征在于：所述试剂盒含有用于特异性扩增33个基因座的33组引物对，33个基因座分别为D3S1358、D13S317、D7S820、D16S539、Penta E、DYS635、DYS456、TPOX、TH01、D2S1338、CSF1PO、DYS385a/b、DYS458、DYS391、D19S433、vWA、D21S11、D18S51、D6S1043、Amelogenin、D8S1179、D5S818、D12S391、FGA、DYS438、DYS393、DYS389I、DYS389II、DYS439、DYS392、Y_GATA_H4及DYS390，33个基因座对应的引物对如下表所示：
    Figure PCTCN2016107578-appb-100001
    Figure PCTCN2016107578-appb-100002
  2. 根据权利要求1所述的人类基因组33个基因座的复合扩增试剂盒，其特征在于：33个基因座的引物在扩增体系中的浓度是：
    Figure PCTCN2016107578-appb-100003
    Figure PCTCN2016107578-appb-100004
  3. 根据权利要求2所述的人类基因组33个基因座的复合扩增试剂盒，其特征在于：每个基因座中的至少一条引物的5’端标记有荧光染料。
  4. 根据权利要求3所述的人类基因组33个基因座的复合扩增试剂盒，其特征在于：33个基因座的引物对使用至少5种不同的荧光染料标记。
  5. 根据权利要求4所述的人类基因组33个基因座的复合扩增试剂盒，其特征在于：33个基因座的引物对使用5种不同的荧光染料标记，根据标记的荧光染料不同分为5组，分别是：
    D3S1358、D13S317、D7S820、D16S539、Penta E和DYS635为第一组；
    DYS456、TPOX、TH01、D2S1338、CSF1PO、DYS385a/b和DYS458为第二组；
    DYS391、D19S433、vWA、D21S11、D18S51、D6S1043为第三组；
    Amelogenin、D8S1179、D5S818、D12S391、FGA、DYS438为第四组；
    DYS393、DYS389I、DYS389II、DYS439、DYS392、Y_GATA_H4及DYS390为第五组。
  6. 根据权利要求5所述的人类基因组33个基因座的复合扩增试剂盒，其特征在于：所述5种不同的荧光染料分别是6-FAM、HEX、TAMRA、ROX和VIG。
  7. 根据权利要求6所述的人类基因组33个基因座的复合扩增试剂盒，其特征在于：还包括反应缓冲液、热启动Taq酶、超纯水、AllelicLadder以及荧光分子量内标，所述反应缓冲液为50mM的Tris-HCl、50mM的KCl、2.0mM的MgCl2、0.2mM的dNTPs和0.8mg/mL的BSA。
  8. 根据权利要求7所述的人类基因组33个基因座的复合扩增试剂盒，其特征在于：聚合酶链式反应的扩增程序为：95℃ 2min；94℃ 30s，61℃ 40s，72℃ 1min，重复15个循环；90℃ 30s，60℃ 1min，65℃ 80s，重复15个循环；60℃ 30min。
  9. 权利要求1～7任一所述的人类基因组33个基因座的复合扩增试剂盒在个体识别、亲子鉴定、嫌疑人家系排查或常染色体数据库及Y染色体数据库中的应用。
PCT/CN2016/107578 2015-12-11 2016-11-29 一种人类基因组33个基因座的复合扩增试剂盒及其应用 WO2017097135A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510918064.4A CN105695569A (zh) 2015-12-11 2015-12-11 一种人类基因组33个基因座的复合扩增试剂盒及其应用
CN201510918064.4 2015-12-11

Publications (1)

Publication Number Publication Date
WO2017097135A1 true WO2017097135A1 (zh) 2017-06-15

Family

ID=56227939

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/107578 WO2017097135A1 (zh) 2015-12-11 2016-11-29 一种人类基因组33个基因座的复合扩增试剂盒及其应用

Country Status (2)

Country Link
CN (1) CN105695569A (zh)
WO (1) WO2017097135A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112176068A (zh) * 2019-07-02 2021-01-05 公安部物证鉴定中心 一种基于29个y-str基因座的复合扩增体系及其使用的引物组合
CN112342297A (zh) * 2019-08-08 2021-02-09 深圳华大法医科技有限公司 用于同时分析多个dip和str位点的复合扩增***、方法、试剂盒及其用途
CN116083595A (zh) * 2022-09-30 2023-05-09 江苏苏博生物医学科技南京有限公司 一种含打拐基因座的33个短串联重复序列复合扩增检测试剂盒及方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105695569A (zh) * 2015-12-11 2016-06-22 广东华美众源生物科技有限公司 一种人类基因组33个基因座的复合扩增试剂盒及其应用
CN106868150B (zh) * 2017-03-09 2019-08-27 广州市刑事科学技术研究所 一种人类常染色体和Y染色体InDel遗传多态性位点复合扩增试剂盒及其应用
CN107201405A (zh) * 2017-06-16 2017-09-26 广州市刑事科学技术研究所 一种硅藻upa基因分析方法及其在法医检测中的应用
CN108251537B (zh) * 2018-01-19 2021-06-22 广东华美众源生物科技有限公司 一种同时扩增人常染色体和y染色体str基因座的荧光标记复合扩增试剂盒及其应用
CN110607374A (zh) * 2019-10-09 2019-12-24 百特元生物科技（北京）有限公司 一种同时扩增人27个str基因座的荧光标记扩增试剂盒及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102725422A (zh) * 2009-09-11 2012-10-10 生命科技公司 Y-染色体str标记的分析
CN104031989A (zh) * 2014-05-16 2014-09-10 无锡中德美联生物技术有限公司 一种人基因组dna26个基因座的复合扩增的试剂盒
CN104946632A (zh) * 2015-05-28 2015-09-30 宁波海尔施基因科技有限公司 一种具有增强鉴别能力的常染色体str基因座荧光标记复合扩增试剂盒及其应用
CN105018623A (zh) * 2015-08-06 2015-11-04 无锡中德美联生物技术有限公司 同时扩增人常染色体和y染色体str基因座的荧光标记复合扩增试剂盒及其应用
CN105695569A (zh) * 2015-12-11 2016-06-22 广东华美众源生物科技有限公司 一种人类基因组33个基因座的复合扩增试剂盒及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104745691A (zh) * 2015-03-02 2015-07-01 无锡中德美联生物技术有限公司 一种同时分析人基因组dna 27个基因座的荧光标记复合扩增的引物组、试剂盒及应用
CN105018597B (zh) * 2015-05-27 2018-04-17 宁波海尔施基因科技有限公司 一种人基因组dna34个基因座的复合扩增试剂盒

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102725422A (zh) * 2009-09-11 2012-10-10 生命科技公司 Y-染色体str标记的分析
CN104031989A (zh) * 2014-05-16 2014-09-10 无锡中德美联生物技术有限公司 一种人基因组dna26个基因座的复合扩增的试剂盒
CN104946632A (zh) * 2015-05-28 2015-09-30 宁波海尔施基因科技有限公司 一种具有增强鉴别能力的常染色体str基因座荧光标记复合扩增试剂盒及其应用
CN105018623A (zh) * 2015-08-06 2015-11-04 无锡中德美联生物技术有限公司 同时扩增人常染色体和y染色体str基因座的荧光标记复合扩增试剂盒及其应用
CN105695569A (zh) * 2015-12-11 2016-06-22 广东华美众源生物科技有限公司 一种人类基因组33个基因座的复合扩增试剂盒及其应用

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112176068A (zh) * 2019-07-02 2021-01-05 公安部物证鉴定中心 一种基于29个y-str基因座的复合扩增体系及其使用的引物组合
CN112176068B (zh) * 2019-07-02 2022-08-02 公安部物证鉴定中心 一种基于29个y-str基因座的复合扩增体系及其使用的引物组合
CN112342297A (zh) * 2019-08-08 2021-02-09 深圳华大法医科技有限公司 用于同时分析多个dip和str位点的复合扩增***、方法、试剂盒及其用途
CN112342297B (zh) * 2019-08-08 2024-01-26 深圳华大法医科技有限公司 用于同时分析多个dip和str位点的复合扩增***、方法、试剂盒及其用途
CN116083595A (zh) * 2022-09-30 2023-05-09 江苏苏博生物医学科技南京有限公司 一种含打拐基因座的33个短串联重复序列复合扩增检测试剂盒及方法
CN116083595B (zh) * 2022-09-30 2023-11-14 江苏苏博生物医学科技南京有限公司 一种含打拐基因座的33个短串联重复序列复合扩增检测试剂盒及方法

Also Published As

Publication number Publication date
CN105695569A (zh) 2016-06-22

Similar Documents

Publication Publication Date Title
WO2017097135A1 (zh) 一种人类基因组33个基因座的复合扩增试剂盒及其应用
US10858645B2 (en) Nucleic acid preparation compositions and methods
RU2708337C2 (ru) Способы и композиции для днк-профилирования
CN107385064B (zh) 一种同时扩增人常染色体snp和str位点的荧光标记复合扩增试剂盒及其应用
ES2909841T3 (es) Nuevo protocolo para preparar bibliotecas de secuenciación
Thompson et al. An overview of DNA typing methods for human identification: past, present, and future
CN105018597B (zh) 一种人基因组dna34个基因座的复合扩增试剂盒
CN107012225B (zh) 一种基于高通量测序的str基因座的检测试剂盒及检测方法
WO2011023131A1 (zh) 20个短串联重复序列的复合扩增试剂盒
KR101667526B1 (ko) 차세대 염기서열분석법을 이용한 인간 객체의 확장 상염색체 str 분석방법
CN109880911A (zh) 25个人类染色体基因座的复合扩增试剂盒及其应用
CN110578009B (zh) 一种包含人类X染色体40个InDel遗传多态性位点的复合扩增检测试剂盒
CN110607374A (zh) 一种同时扩增人27个str基因座的荧光标记扩增试剂盒及其应用
CN110863056A (zh) 一种人类dna精准分型的方法、试剂和应用
CN104131072A (zh) 一种对未知检材进行个体识别和亲权鉴定的方法和***
CN113416791B (zh) 同时扩增人25个str基因座的特异性扩增引物组、荧光标记扩增试剂盒及应用和方法
CN110157812A (zh) 一种同时检测常染色体和y染色体str基因座的复合扩增试剂盒
CN112852971A (zh) 一种同时扩增人44个y-str基因座的引物组、试剂盒及其应用
CN106086223B (zh) 一种基于zna引物的str分型试剂盒
CN108251537B (zh) 一种同时扩增人常染色体和y染色体str基因座的荧光标记复合扩增试剂盒及其应用
EP3315612B1 (en) Set of primers and method for detecting and identifying mussel species of the genus mytilus
CN111575386B (zh) 一种检测人y-snp基因座的荧光复合扩增试剂盒及应用
CN112852973A (zh) 一种同时扩增人13个str基因座的引物组、试剂盒及其应用
CN109852704B (zh) 一种同时检测32个y染色体基因座的复合扩增试剂盒
CN112266952A (zh) 针对疑难检材的补充str位点复合扩增试剂盒及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16872326

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 12.11.2018)

122 Ep: pct application non-entry in european phase

Ref document number: 16872326

Country of ref document: EP

Kind code of ref document: A1