WO2017097135A1 - Kit d'amplification composite pour 33 loci génétiques du génome humain et son application - Google Patents

Kit d'amplification composite pour 33 loci génétiques du génome humain et son application Download PDF

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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
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loci
human genome
amplification kit
composite amplification
kit
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PCT/CN2016/107578
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Chinese (zh)
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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
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广东华美众源生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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.

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Abstract

La présente invention concerne un kit d'amplification composite pour 33 loci génétiques du génome humain. Les loci génétiques comprennent 18 loci génétiques A-STR, 14 loci génétiques Y-STR, et un locus génétique de l'Amélogénine. Le kit amplifie et détecte de manière synchrone 33 loci génétiques au moyen d'un tube unique, ce qui permet de réaliser une amplification et une détection synchrones d'euchromosome et de chromosome Y dans une expérience.
PCT/CN2016/107578 2015-12-11 2016-11-29 Kit d'amplification composite pour 33 loci génétiques du génome humain et son application WO2017097135A1 (fr)

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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位点的复合扩增系统、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

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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

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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

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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位点的复合扩增系统、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位点的复合扩增系统、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

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