WO2016188331A1 - 一种人类基因组dna 34个基因座的复合扩增试剂盒 - Google Patents

一种人类基因组dna 34个基因座的复合扩增试剂盒 Download PDF

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WO2016188331A1
WO2016188331A1 PCT/CN2016/082019 CN2016082019W WO2016188331A1 WO 2016188331 A1 WO2016188331 A1 WO 2016188331A1 CN 2016082019 W CN2016082019 W CN 2016082019W WO 2016188331 A1 WO2016188331 A1 WO 2016188331A1
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seq
kit
nos
loci
str
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李政
张兹钧
余丁
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宁波海尔施基因科技有限公司
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    • 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
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    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention belongs to the field of biotechnology autosomal and Y chromosome typing and identification, and relates to a PCR amplification kit, in particular to a single tube simultaneous detection of six-color fluorescent labeled complex amplification of human genome autosomal and Y chromosome loci.
  • a short tandem repeat is a type of DNA sequence with a length polymorphism formed by tandem repeats of 2-6 bases as a core unit in the human genome. The number of core units varies and the number of repeats is different. It constitutes the genetic polymorphism of STR. STR is widely distributed and multiplied, accounting for about 10% of the human genome. It contains a huge amount of information. Different sequences can produce hundreds of millions of genotype combinations, and each combination has a very low frequency in the population. It has a very high ability to identify individuals, so it is often used as a genetic marker for forensic individual identification and phylogenetic identification in DNA analysis technology. It is also the mainstream technology for DNA database establishment.
  • the fragment of the STR locus is small and easy to amplify, suitable for testing trace and degradation samples, and the amplification conditions of each locus are similar and can be combined and amplified, so it is sensitive, accurate, rapid, and has a large amount of information. . Because of these advantages, the typing and screening of STR loci have been widely used in anthropology, medical genetics and forensic science and related fields at home and abroad.
  • the human Y chromosome is a small proximal centromere chromosome composed of a long arm and a tiny short arm.
  • the Y chromosome except for the autosomal region, does not undergo exchange and recombination in meiosis, and is uniploidally transmitted downward, showing the paternal genetic characteristics, while the sequence variation is completely caused by the cumulative mutation, not caused by recombination. .
  • the Y chromosome STR locus (Y-STR) genetic marker has been widely used as a tool for forensic identification, paternity testing, identification of missing persons, human migration evolution studies, history and family evolution studies, etc. Multiple areas.
  • Y-STR loci More than 400 Y-STR loci have been discovered, and the 9-European smallest haplotype loci are commonly used in the Y-STR locus, including DYS19, DYS385a/b, DYS389I/II, DYS390, DYS392, DYS393. And two genetic loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including DYS438 and DYS439.
  • SWGDAM Scientific Working Group on DNA Analysis Methods
  • the first Y-STR kit is Y-PLEXTM6, developed by ReliaGene Technologies in 2001. It can amplify DYS19, DYS389II, DYS390, DYS391, DYS393, DYS385a/b; 2002 ReliaGene Technologies Y-PLEXTM5, which can amplify DYS389I/II, DYS439, DYS438, and DYS392, was developed.
  • ReliaGene Technologies introduced Y-PLEXTM12, which integrates all Y-PLEXTM6 and Y-PLEXTM5 loci.
  • PowePlex Y23 kit is the foreign kit that can synthesize the most amplified Y-STR on the market.
  • the development of the domestic Y-STR kit is relatively late.
  • the AGCU Y24 STR fluorescence detection kit from Jiangsu Zhongde Meilian Co., Ltd. contains 24 Y-STR loci including DYS391, DYS389I/II, DYS439, DYS438, DYS449, DYS456, DYS458, DYS437, DYS635, DYS448. , DYS527a/b, GATA H4, DYS447, DYS19, DYS392, DYS522, DYS393, DYS388, DYS390, DYS385a/b and DYS444.
  • Patent CN101144774 discloses a fluorescently labeled complex amplification system for simultaneously analyzing multiple loci of human genomic DNA for detecting the following 21 autosomal loci: TPOX, D3S1358, D5S818, FGA, CSF1PO, D8S1179, D7S820, TH01 , VWA, D13S317, D16S539, D18S51, D21S11, D12S391, D19S433, D1S1656, D2S1338, D6S1043, PentaE, PentaD, AMEL.
  • the patent provides a typing result for 20 autosomal loci and a sex locus, and is a five-color fluorescence detection system.
  • autosomal typing and Y chromosome testing are generally separated.
  • DYS391 was added to the autosomal assay at most. Because DYS391 is poorly polymorphic, it is mainly used to assist gender determination. If the autosomal locus and the more polymorphic Y chromosome locus can be detected simultaneously, the Y chromosome locus information can be used for the rapid investigation of the suspect, and the suspect can be determined based on the information of the frequently stained locus.
  • the present invention is directed to the above technical problems.
  • 16 loci AMEL, TPOX, D3S1358, D5S818, FGA, CSF1PO, D8S1179, D7S820, TH01, VWA, D13S317, D16S539, D18S51, D21S11, D2S1338 are selected.
  • D19S433 so that the locus is the same as the ABI (Applied Biosystems, USA) Identifiler kit locus, and 18 Y chromosome loci are added, including the Afile (Applied Biosystems, USA) Yfiler kit.
  • 17 loci and an insertion deletion polymorphism locus Y-indel are selected from the ABI (Applied Biosystems, USA).
  • One of the objects of the present invention is to provide a fluorescent-labeled STR complex amplification test system for performing individual recognition and paternity testing by complex amplification of 34 loci, and simultaneously detecting 16 autosomal loci and 17 Y chromosome genes. Block and a sex locus. It involves the detection of genetic markers with polymorphisms in the human locus.
  • the present invention simultaneously adds 17 Y loci DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS438, DYS389I, DYS19, GATA in the most widely used Yfiler kit. -H4, DYS389II, DYS448 and an insertion deletion polymorphism locus Y-indel on the Y chromosome.
  • the gene diversity (GD) values of all loci except DYS391 were above 0.6, and DYS391 was the latest CODIS recommended locus in the United States.
  • the ability to add the Y locus is much better than the patent CN101144774.
  • kits for determining a composite amplification of 34 loci of human genomic DNA Based on the determination of the above loci, the present invention provides a kit for complex amplification of 34 loci of human genomic DNA, and comprises a specific oligonucleotide amplification primer pair of the following 34 STR loci: Y-indel , DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448, TPOX, D3S1358, D5S818, FGA, CSF1PO, D8S1179, D7S820 , TH01, VWA, D13S317, D16S539, D18S51, D21S11, D2S1338, D19S433 and A
  • each pair of primers has a certain primer sequence for amplifying the corresponding locus, and the corresponding relationship is as shown in Table 2:
  • each of the STR loci is amplified using a pair of primers located on either side of the core repeat region of the locus, wherein each of the pair of primers has a fluorescent dye label on the 5' end of the primer.
  • the invention adopts a reasonable arrangement of individual loci and preferably a series of fluorescent dyes with high fluorescence intensity, and the labeling methods are: Y-indel, D3S1358, TH01, D21S11, D18S51, D19S433, DYS439 adopt FAM label; DYS438, DYS389I , DYS448, DYS389II, DYS19, GATA-H4, DYS458 use VIC mark; AMEL, D5S818, D13S317, D7S820, D16S539, CSF1PO, D2S1338 use NED mark; DYS437, VWA, D8S1179, TPOX, FGA, DYS456 use ROX mark; DYS393, DYS391, DYS390, DYS635, DYS392, DYS385a/b were labeled with AF594; the internal standard was selected with orange fluorescent label and the fluorescent label was
  • the method for detecting an amplification product of the present invention is carried out by using a multi-channel or single-channel capillary electrophoresis genetic analyzer; the template determined by the present invention includes human blood, blood mark, semen, saliva, body fluid, hair, muscle or tissue organ, and Direct amplification filter paper, FTA card, cotton wool, buccal swab and other materials can be used.
  • the template determined by the present invention includes human blood, blood mark, semen, saliva, body fluid, hair, muscle or tissue organ, and Direct amplification filter paper, FTA card, cotton wool, buccal swab and other materials can be used.
  • the kit for comprehensive amplification of 34 loci of human genomic DNA comprises nuclease-free water, PCR buffer, primer mixture, allelic standard, positive control, molecular weight internal standard and spectral correction A standard, said allelic ladder is a mixture of all of the different genotypes of a locus contained in a kit in a population of numbers, the spectrally correcting standards being fluorescent PCR amplification products of different sizes.
  • the kit for complex amplification of 34 loci of human genomic DNA comprises nuclease-free water, PCR Master Mix, SureID Compass Primer Mix, SureID Compass Allelic ladder mixture, internal standard Size-500 .
  • the PCR Master Mix used in the present invention has been subjected to a series of optimization experiments to make the product compatible with all common types of materials on the market including whatman FTA card, whatman saliva card, blood filter paper, Bokun FTA card, Bo Kun's saliva card, hair, oral exfoliated cells, DNA extraction and other samples, which have not been done in domestic or even foreign kits, in addition, this improved buffer can greatly improve the amplification efficiency It can effectively shorten the time of adenylation at the end of the product, and can improve the amplification efficiency of the long fragment and improve the balance of the product.
  • Its main components are: DMSO, Tris-buffer, potassium chloride, ammonium sulfate and the like.
  • kits provided by the present invention detect more gene loci than the domestic and international simple autosomal detection kits and Y chromosome detection kits, thereby greatly improving the cumulative individual recognition of the system and Cumulative non-parent exclusion rate, generally improve the individual's discriminating power.
  • Figure 1 is a map of the allelic ladder of the fluorescent-labeled complex amplification assay system of 34 loci;
  • Figure 2 is a partial view of the male standard 9948
  • Figure 3 is a classification diagram of the amplified database
  • Figure 4a is a fragmentation diagram of the STRtyper-21G kit for detecting a boy to be examined
  • Figure 4b is a fragmentation diagram of the STRtyper-21G kit for detecting a man to be examined
  • Figure 5a is a fragmentation diagram of the SureID compass kit for detecting a boy to be examined
  • Figure 5b is a fragmentation diagram of the SureID compass kit for detecting a man to be examined.
  • the present invention simultaneously adds 17 Y loci DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS438, DYS389I, DYS19, GATA in the most widely used Yfiler kit. -H4, DYS389II, DYS448 and an insertion deletion polymorphism locus Y-indel on the Y chromosome.
  • the GD values of all loci except DYS391 were above 0.6, and DYS391 was the latest CODIS recommended locus in the United States.
  • the ability to add the Y locus is much better than the patent CN101144774.
  • the invention has identified and selected fluorescent dyes, and selected six fluorescent labels of blue, green, yellow, red, purple and orange to construct a six-color fluorescent combination scheme. Based on the determination of the 6-color fluorescence combination scheme, the gene locus combination method and the fluorescent label type were designed through a large number of repeated experiments. From the perspective of production cost and amplification efficiency of primers at each locus, 34 loci were divided into 5 groups, labeled with FAM, VIC, NED, ROX, AF594, and the molecular weight internal standard was labeled with the sixth color orange fluorescent dye Atto. 633 is marked.
  • a preferred method for labeling fluorescent dyes that has been finalized by screening is: Y-indel, D3S1358, TH01, D21S11, D18S51, D19S433, DYS439 using FAM labeling; DYS438, DYS389I, DYS448, DYS389II, DYS19, GATA-H4, DYS458 VIC mark; AMEL, D5S818, D13S317, D7S820, D16S539, CSF1PO, D2S1338 use NED mark; DYS437, VWA, D8S1179, TPOX, FGA, DYS456 use ROX mark; DYS393, DYS391, DYS390, DYS635, DYS392, DYS385a/b AF594; the internal standard was selected with orange fluorescent label and the fluorescent label was Atto 633. This combination of loci allows 34 bases to be achieved with only six fluorescent labels. Simul
  • the PCR Master Mix includes: DMSO 10 mM, Tris-buffer 125 mM, potassium chloride 125 mM, ammonium sulfate 65 mM, which can achieve compatible amplification of various common materials in the market.
  • the SureID Compass Primer Mix includes all primers for amplifying 34 loci (see Table 2 for concentration), deoxynucleotide triphosphate (dATP, dGTP, dTTP, dCTP) 7.5 mM, Taq 5 U/ ⁇ l, magnesium chloride 7.5 mM , BSA 2.5mg/ml.
  • Control DNA 9948 was used as a positive control and was human genomic DNA purchased from Suzhou Xinhai Biotechnology Co., Ltd.
  • the Allelic Ladder allelic ladder is a mixture of all the different genotypes of the locus contained in the kit in a certain population, from Ningbo Haiershi Gene Technology Co., Ltd.
  • the Size-500 orange fluorescent molecular weight internal standard is a series of plasmids used to calibrate fragments of a certain size, from Ningbo Haiershi Gene Technology Co., Ltd.
  • the spectral calibration standard is a fluorescent PCR amplification product of six different size fragments from Ningbo Haiershi Gene Technology Co., Ltd.
  • step temperature time 1 95 ° C 5 minutes 2 94°C 10 seconds 3 61 ° C 1 minute 4 70 ° C 30 seconds 5 N/A Repeat 2-4 steps 27 times (28 times in total) 6 60 ° C 20 minutes 7 4 ° C Continuous: until the PCR product is collected
  • a sample mixture ⁇ (1 ⁇ L Size-500 + 12 ⁇ L deionized formamide) ⁇ was composed of deionized formamide and a molecular weight internal standard (Size-500) in the system.
  • the analysis was carried out by ABI 3500 Genetic Analyzer. The specific analysis parameters were injection voltage: 1.2kv, injection time: 15s, and electrophoresis time: 1210-1500s. .
  • Sensitivity analysis After the positive control is diluted by a certain copy number ratio, it is detected by PCR amplification and capillary electrophoresis until no signal is detected.
  • the copy number is the lowest detection line, which is the sensitivity of the kit. The highest sensitivity detects DNA samples as low as 0.125 ng.
  • the fluorescent labeling complex amplification test system of 34 loci in the present invention tests pigs, dogs, sheep, ducks, chickens, mice, cattle, Escherichia coli, etc., without specific amplification peaks, indicating that the system has Species specificity.
  • the kit provided by the invention is used for simultaneously establishing an autosomal database and a Y chromosome database, Proceed as follows:
  • the sample in this case is a filter paper blood sample, which is directly amplified, so it is only necessary to use a 1.2 mm puncher for punching as a test template;
  • Amplification detection According to Examples 2 to 4, fluorescent labeling, PCR amplification and genetic analyzer detection were carried out, and a kit for amplifying primer pairs of a specific oligonucleotide containing 34 STR loci was used, and the detection spectrum was selected. See Figure 3, the results of the classification are shown in the following table:
  • kits provided by the invention can satisfy the establishment of the autonomic library and the establishment of the Y chromosome database in the establishment of the forensic DNA database, which greatly reduces the repeated experiments and data analysis, and saves time.
  • Example 6 The gene locus complex amplification kit disclosed in Patent CN101144774 is separately applied. (STRtyper-21G kit) and the kit provided by the present invention (SureID compass kit) for single parental identification
  • the sample of single parent paternity test in this embodiment is provided by a pair of fathers and sons of Ningbo City;
  • the sample in this case is a filter paper blood sample, which is directly amplified, so it is only necessary to use a 1.2 mm puncher for punching as a test template;
  • the present invention has sufficient number of autosomal loci, and more Y chromosome loci greatly improve resolution and elimination rate, and exhibit great superiority in single parental paternity identification.
  • the identification of single parent-child relationship it is necessary to detect the autosomal locus and the Y chromosome locus.
  • the current practice is to detect it by an autosomal locus detection kit and a Y chromosome locus detection kit. In this example, based on the results of 21 autosomal loci detection, the father-son relationship cannot be identified or excluded; and the increased Y locus ultimately excludes the parent-child relationship.
  • the two kits required before namely, the autosomal locus detection kit and the Y chromosome locus detection kit are integrated into one kit, and only one PCR amplification and electrophoresis detection can be performed. Get more information, improve detection efficiency and individual discernment.
  • the test results of the paternity test kit are shown in Table 4 below.

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Abstract

本发明提供一种人类基因组DNA 34个基因座的复合扩增试剂盒，所述34个STR基因座为：DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448、TPOX、D3S1358、D5S818、FGA、CSF1PO、D8S1179、D7S820、TH01、VWA、D13S317、D16S539、D18S51、D21S11、Y-indel、D2S1338、D19S433及Amelogenin。

Description

一种人类基因组DNA 34个基因座的复合扩增试剂盒 技术领域
本发明属于生物技术常染色体和Y染色体分型和鉴定领域，涉及一种PCR扩增试剂盒，尤其涉及一种单管同时检测人类基因组常染色体及Y染色体基因座的六色荧光标记复合扩增试剂盒以及该试剂盒在司法鉴定领域中的应用。
背景技术
短串联重复序列(short tandem repeat，STR)是人类基因组中由2-6个碱基作为核心单位串联重复形成的一类具有长度多态性的DNA序列，其核心单位的数目变化和重复次数不同构成了STR的遗传多态性。STR分布广，数目多，约占人类基因组的10％，所包含的信息量巨大，不同的序列可以产生数以亿计的基因型组合，而每一种组合在群体中出现的频率都非常低，具有极高的个体鉴定能力，所以常作为遗传标记而被用于DNA分析技术中法医个体识别、亲缘关系鉴定，同时也是DNA数据库建立的主流技术。同时，STR基因座的片段小，容易扩增，适宜于检验微量和降解检材，而且各基因座的扩增条件相似而能够复合扩增，因而具有灵敏、准确、快速、信息量大等优点。由于这些优点STR基因座的分型研究与筛选早已在国内外人类学、医学遗传学和法医学及各相关领域中得到了广泛的应用。
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，在减数分裂中不发生交换重组，呈单倍型独立向下传递，表现出父系遗传特征，同时序列的变异完全由累积的突变所致，并非重组引起。由于Y染色体的这些特点，Y染色体STR基因座(Y-STR)遗传标记作为一种工具已经被广泛应用于法医鉴定、亲权鉴定、失踪人员鉴定、人类迁移进化研究、历史和家系进化研究等多个领域。已发现的Y-STR基因座已有400多个，常用来检测的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、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。
在法医鉴定与亲子鉴定领域，DNA分析主要依赖商业化的试剂盒进行。第一个Y-STR试剂盒是由ReliaGene Technologies在2001年研发的Y-PLEXTM6，可复合扩增DYS19、DYS389II、DYS390、DYS391、DYS393、DYS385a/b；2002年ReliaGene Technologies 又研发了可复合扩增DYS389I/II、DYS439、DYS438、DYS392的Y-PLEXTM5，到2003年9月ReliaGene Technologies推出了整合了Y-PLEXTM6和Y-PLEXTM5所有基因座在内的Y-PLEXTM12。同年10月，Promega公司推出了PowerPlex Y试剂盒，可复合扩增12个基因座，其中包括DYS19、DYS385a/b、DYS389I/II、DYS390、DYS391、DYS392、DYS393、DYS437、DYS438、DYS439。2004年秋，Applied Biosystems公司(简称AB公司)发布了包含17个Y-STR基因座的yfilerTM试剂盒，其在PowerPlex Y的基础上增加了另外5个具有高度多态性的Y-STR基因座，包括DYS456、DYS458、DYS635、GATA-H4、DYS448。2012年秋，Promega公司推出了PowerPlex Y23试剂盒，在兼容了yfilerTM试剂盒的17个基因座外又额外增加了6个新基因座包括DYS549、DYS481、DYS533、DYS570、DYS576、DYS643，进一步提高了试剂盒的分辨率。目前PowePlex Y23试剂盒是目前市面上可复合扩增Y-STR最多的国外试剂盒。国内Y-STR试剂盒的发展相对较晚，目前主要有北京基点认知公司的Goldeneye 20Y试剂盒，可复合扩增20个Y-STR基因座，除包含yfilerTM试剂盒的17个基因座外新增加了DYS460、DYS447、DYS388。此外还有江苏中德美联公司推出的AGCU Y24 STR荧光检测试剂盒，包含了24个Y-STR基因座包括DYS391、DYS389I/II、DYS439、DYS438、DYS449、DYS456、DYS458、DYS437、DYS635、DYS448、DYS527a/b、GATA H4、DYS447、DYS19、DYS392、DYS522、DYS393、DYS388、DYS390、DYS385a/b以及DYS444。
随着法医DNA分型技术的发展，目前已经能对类似血痕、精斑、唾液斑、毛发、骨骼等进行DNA分型；同时，随着PCR技术的发展，多重扩增体系可检测的基因座数量也越来越多。专利CN101144774公开了一种同时分析人类基因组DNA的多个基因座的荧光标记复合扩增系统，用于检测如下21个常染色体基因座：TPOX、D3S1358、D5S818、FGA、CSF1PO、D8S1179、D7S820、TH01、VWA、D13S317、D16S539、D18S51、D21S11、D12S391、D19S433、D1S1656、D2S1338、D6S1043、PentaE、PentaD、AMEL。该专利提供了对20个常染色体基因座和一个性别基因座的分型结果，为五色荧光检测系统。目前在法医检测中，常染色体分型与Y染色体检测一般都是分开的。最多在常染色体检测中加入了DYS391。因为DYS391多态性差，主要用于辅助性别判定。如果能同时检测常染色体基因座及多态性更高的Y染色体基因座，则既能用Y染色体基因座信息用于嫌疑人的快速排查，又能根据常染色基因座信息确定嫌疑人。
发明内容
本发明针对以上技术问题，在发明CN101144774方案的基础上，选择了16个基因座AMEL、TPOX、D3S1358、D5S818、FGA、CSF1PO、D8S1179、D7S820、TH01、VWA、D13S317、D16S539、D18S51、D21S11、D2S1338、D19S433，这样使得基因座和ABI(Applied Biosystems,USA)公司的Identifiler试剂盒基因座一样，同时加入了18个Y染色体基因座，其中包含了ABI(Applied Biosystems,USA)公司的Yfiler试剂盒的17个基因座以及一个插入缺失多态性基因座Y-indel。
本发明的目的之一在于：提供一种通过复合扩增34个基因座来进行个体识别和亲子鉴定的荧光标记STR复合扩增检验系统，同时检测16个常染色体基因座、17个Y染色体基因座及一个性别基因座。涉及检测人基因座中具有多态性的遗传标记。
为了实现本发明的目的，采用的技术方案为：
(1)确定基因座。在专利CN101144774的基础上，选择与ABI公司Identifiler试剂盒相同的16个基因座，通过对这些基因座进行频率调查分别计算个体识别能力(DP)、杂合度(H)、非父排除率(PE)等数据，表明在所述16个STR基因座中，除TH01、TPOX基因座外，其余各基因座的DP值均接近0.9，H均大于0.7，PE值大都在0.5以上，这表明它们在法医学上有较好的应用价值。保持现有的常染色STR，增加一定个数的Y-STR，对亲缘关系的鉴定，家系排查，估计可能的人群来源有巨大的作用。此外，本发明同时添加了使用最广泛的Yfiler试剂盒里的17个Y基因座DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448以及Y染色体上的一个插入缺失多态性基因座Y-indel。除DYS391之外其余基因座的基因多样性(gene diversity,GD)值均在0.6以上，DYS391为美国最新CODIS推荐的基因座。添加Y基因座的排查能力大大优于专利CN101144774。
(2)确定一种人类基因组DNA 34个基因座的复合扩增的试剂盒。基于以上基因座的确定，本发明提供一种人类基因组DNA 34个基因座的复合扩增的试剂盒，同时包含下列34个STR基因座的特异性寡核苷酸扩增引物对：Y-indel、DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448、TPOX、D3S1358、D5S818、FGA、CSF1PO、D8S1179、D7S820、TH01、VWA、D13S317、D16S539、D18S51、D21S11、D2S1338、D19S433及Amelogenin。所述34个基因座的具体信息见表1。
表1：34个基因座的相关信息
Figure PCTCN2016082019-appb-000001
Figure PCTCN2016082019-appb-000002
进一步地，每对引物具有一定的引物序列，其用于扩增相应的基因座，其对应关系如表2所示：
表2：各基因座对应的引物序列及其浓度
Figure PCTCN2016082019-appb-000003
Figure PCTCN2016082019-appb-000004
优选地，本发明的试剂盒中所述各基因座对应的引物浓度如表2所示，这样测出的结果中无非特异性峰。进一步地，所述的各STR基因座采用位于该基因座核心重复区两侧的一对引物扩增，其中每对引物中有一条引物的5’端进行荧光染料标记。
本发明通过对各个基因座的合理排列并优选了一系列荧光强度高的荧光染料进行标记，标记方法为：Y-indel、D3S1358、TH01、D21S11、D18S51、D19S433、DYS439采用FAM标记；DYS438、DYS389I、DYS448、DYS389II、DYS19、GATA-H4、DYS458采用VIC标记；AMEL、D5S818、D13S317、D7S820、D16S539、CSF1PO、D2S1338采用NED标记；DYS437、VWA、D8S1179、TPOX、FGA、DYS456采用ROX标记；DYS393、 DYS391、DYS390、DYS635、DYS392、DYS385a/b采用AF594标记；内标选用橙色荧光标记，荧光标记物为Atto 633。
本发明检测扩增产物的方法为采用多道或单道毛细管电泳遗传分析仪进行测定；本发明所测定的模板包括人的血液、血痕、精液、唾液、体液、毛发、肌肉或组织器官，并且可以进行直接扩增滤纸、FTA卡、棉絮、口腔拭子等检材。
本发明提供的人类基因组DNA 34个基因座的复合扩增的试剂盒包括无核酸酶水、PCR缓冲液、引物混合液、等位基因分型标准品、阳性对照品、分子量内标以及光谱校正标准物，所述等位基因分型标准品是试剂盒所包含的基因座在一定数量人群中的所有不同基因分型的混合物，所述光谱校正标准物是不同大小的荧光PCR扩增产物。
在一个实施方案中，本发明提供的人类基因组DNA 34个基因座的复合扩增的试剂盒包括无核酸酶水、PCR Master Mix、SureID Compass Primer Mix、SureID Compass Allelic ladder混合物、内标Size-500。值得一提的是本发明所采用的PCR Master Mix经过一系列的优化实验使得本产品可以兼容市面上所有常见的检材类型包括whatman FTA卡、whatman唾液卡、血滤纸、博坤FTA卡、博坤唾液卡、头发、口腔脱落细胞、提取DNA等各种检材，这在国内甚至国外的试剂盒也尚未做到，除此之外，该种改良后的缓冲液能极大地提高扩增效率，有效缩短产物末端腺苷酰化的时间，并能够提高长片段的扩增效率，改善了产品的均衡性。其主要成分有：DMSO、Tris-buffer、氯化钾、硫酸铵等。
综上所述，本发明所提供的试剂盒所检测的基因座数量多于市面上国内外单纯的常染色体检测试剂盒和Y染色体检测试剂盒，从而极大地提高了系统的累积个体识别力和累积非父排除率，总体上提高个体的鉴别力。
附图说明
图1为34个基因座的荧光标记复合扩增检验系统等位基因分型标准物图谱；
图2为男性标准品9948的分型图；
图3为扩增建库样本的分型图；
图4a为STRtyper-21G试剂盒检测待检男孩的分型图；
图4b为STRtyper-21G试剂盒检测待检男子的分型图；
图5a为SureID compass试剂盒检测待检男孩的分型图；
图5b为SureID compass试剂盒检测待检男子的分型图。
具体实施方式
为了更好地理解本发明的内容，下面结合具体实施例和附图作进一步说明。应理解，这些实施例仅用于对本发明进一步说明，而不用于限制本发明的范围。此外应理解，在阅读了本发明所述的内容后，本领域技术人员对本发明作出的一些非本质的改动或调整仍属于本发明的保护范围。
实施例1 基因座的确定
在专利CN101144774的基础上，选择与ABI公司Identifiler试剂盒相同的16个基因座，通过对这些基因座进行频率调查分别计算个体识别能力(DP)、杂合度(H)、非父排除率(PE)等数据，表明在所述16个STR基因座中，除TH01、TPOX基因座外，其余各基因座的DP值均接近0.9，H均大于0.7，PE值大都在0.5以上，这表明它们在法医学上有较好的应用价值。保持现有的常染色体STR，增加一定个数的Y-STR，对亲缘关系的鉴定、家系排查、可能的人群来源的估计都有巨大的作用。此外，本发明同时添加了使用最广泛的Yfiler试剂盒中的17个Y基因座DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448以及Y染色体上的一个插入缺失多态性基因座Y-indel。除DYS391之外其余基因座的GD值均在0.6以上，DYS391为美国最新CODIS推荐的基因座。添加Y基因座的排查能力大大优于专利CN101144774。
实施例2 荧光标记复合扩增体系的基因座组合方案设计
本发明对荧光染料进行了鉴别、遴选，选用了蓝、绿、黄、红、紫、橙六种荧光标记物，构建了6色荧光组合方案。在确定6色荧光组合方案的基础上，通过大量反复实验，设计出基因座组合方式以及荧光标记类型。从生产成本及各基因座引物扩增效率等方面考虑，将34个基因座分成5组，使用FAM、VIC、NED、ROX、AF594分组标记，分子量内标用第6种颜色橙色的荧光染料Atto 633进行标记。经过筛选最终确定的一种优选的荧光染料标记的方法为：Y-indel、D3S1358、TH01、D21S11、D18S51、D19S433、DYS439采用FAM标记；DYS438、DYS389I、DYS448、DYS389II、DYS19、GATA-H4、DYS458采用VIC标记；AMEL、D5S818、D13S317、D7S820、D16S539、CSF1PO、D2S1338采用NED标记；DYS437、VWA、D8S1179、TPOX、FGA、DYS456采用ROX标记；DYS393、DYS391、DYS390、DYS635、DYS392、DYS385a/b采用AF594；内标选用橙色荧光标记，荧光标记物为Atto 633。这种基因座组合方式使得仅需标记六种荧光就可以实现34个基 因座同时检测分析。
实施例3 扩增基因座及其产物检测的实验过程
本发明提供的人类基因组DNA 34个基因座的复合扩增的试剂盒包括：
1)PCR Master Mix
2)SureID Compass Primer Mix
3)Control DNA 9948
4)SureID Compass Allelic Ladder等位基因分型标准品
5)Size-500橙色荧光分子量内标
6)光谱校正标准物
所述PCR Master Mix包括：DMSO 10mM、Tris-buffer 125mM、氯化钾125mM、硫酸铵65mM，可以实现兼容扩增市面常见各种检材。
所述SureID Compass Primer Mix包括扩增34个基因座的所有引物(浓度见表2)、三磷酸脱氧核苷酸(dATP、dGTP、dTTP、dCTP)7.5mM、Taq酶5U/μl、氯化镁7.5mM、BSA 2.5mg/ml。
所述Control DNA 9948作为阳性对照品，是人类基因组DNA，购自苏州新海生物科技股份有限公司。
所述Allelic Ladder等位基因分型标准品是试剂盒所包含的基因座在一定数量人群中的所有不同基因分型的混合物，来自宁波海尔施基因科技有限公司。
所述Size-500橙色荧光分子量内标是一系列用于标定一定大小片段的质粒，来自宁波海尔施基因科技有限公司。
所述光谱校正标准物是6种不同大小片段的荧光PCR扩增产物，来自宁波海尔施基因科技有限公司。
1、反应体系的配置
组分 体积
无核酸酶水(无RNA酶/DNA酶超纯水Dnase/Rnase Free) 补足至25.0μL
PCR Master Mix 12.5μL
SureID Compass Primer Mix 6.25μL
Control DNA 9948 1ng
总 25μL
2、扩增热循环实验方案
1)将PCR扩增管置于热循环仪上；
2)选择下表3推荐的程序进行扩增；
3)扩增后的样品应避光保存；
表3：热循环仪的扩增程序
步骤 温度 时间
1 95℃ 5分钟
2 94℃ 10秒钟
3 61℃ 1分钟
4 70℃ 30秒钟
5 N/A 重复2-4步骤27次(共28次)
6 60℃ 20分钟
7 4℃ 持续:直至收取PCR产物
3、扩增产物在ABI3500遗传分析仪上检测
由去离子甲酰胺与系统中分子量内标(Size-500)组成上样混合物{(1μL Size-500+12μL去离子甲酰胺)×(进样数)}。将9μL上样混合物与1μL扩增产物或系统中等位基因分型标准物(Allelic ladder)混合，避免产生气泡，尽快电泳。用ABI 3500遗传分析仪检测分析，具体分析参数为进样电压：1.2kv，进样时间：15s，电泳时间1210-1500s。。
实施例4 检测试剂盒灵敏度、特异性分析
灵敏度分析：将阳性对照按一定拷贝数倍比稀释后，经PCR扩增和毛细管电泳检测直至检测不到信号，该拷贝数即为最低检测线，也就是试剂盒的灵敏度。最高灵敏度可检测到低至0.125ng的DNA样品。
特异性分析：本发明中34个基因座的荧光标记复合扩增检验系统检验猪、狗、羊、鸭、鸡、鼠、牛、大肠杆菌等，没有出现特异性扩增峰，表明该系统具有种属特异性。
上述实施例并非是对本发明的限制，本发明也并不限于上述实施例。本技术领域的普通技术人员在本发明的实质范围内，做出的变化、改型、添加或替换，也应属于本发明的保护范围。
实施例5 本发明所提供的试剂盒在DNA数据库建设中的应用
将本发明所提供的试剂盒用于同时进行常染色体数据库和Y染色体数据库的建立， 步骤如下：
1.收集公安部门建库样本：样本由某公安部门提供；
2.待检检材的处理：本案中的检材为滤纸血样，采用直接扩增，故只需使用1.2mm的打孔器进行打孔作为检测模板；
3.扩增检测：按照实施例2～4进行荧光标记、PCR扩增和遗传分析仪检测，选用同时包含34个STR基因座的特异性寡核苷酸扩增引物对的试剂盒，检测图谱见图3，分型结果见下表：
常染色体STR基因座 基因型 Y-STR基因座 基因型
D3S1358 15/17 Y-indel 2
TH01 6/9.3 DYS439 12
D21S11 29/30 DYS438 11
D18S51 15/18 DYS389I 13
D19S433 13/14 DYS448 19
D5S818 11/13 DYS389II 31
D13S317 11 DYS19 14
D16S539 11 GATA-H4 12
CSF1PO 10/11 DYS458 18
D2S1338 23 DYS437 15
VWA 17 DYS456 17
D8S1179 12/13 DYS393 13
TPOX 8/9 DYS391 10
FGA 24/26 DYS390 24
D7S820 11 DYS635 23
    DYS392 13
    DYS385a/b 11/14
结果显示：本发明所提供的试剂盒在法医DNA数据库的建立方面可满足同时进行常染色体库建立和Y染色体数据库的建立，极大地减少了重复实验和数据分析，节约了时间。
实施例6 分别应用专利CN101144774中公开的基因座复合扩增试剂盒 (STRtyper-21G试剂盒)及本发明所提供的试剂盒(SureID compass试剂盒)进行单亲亲子鉴定
将专利CN101144774中公开的试剂盒和本发明所提供的试剂盒用于单亲亲子鉴定，测定步骤如下：
1.收集亲子鉴定案件中的血样：本实施例中的单亲亲子鉴定样本由宁波市的一对父子所提供；
2.待检检材的处理：本案中的检材为滤纸血样，采用直接扩增，故只需使用1.2mm的打孔器进行打孔作为检测模板；
3.结论：分别应用STRtyper-21G及Sure ID compass荧光标记复合扩增检测系统对本实施例中的单亲父子进行亲子鉴定，其中所使用的STRtyper-21G复合扩增系统中有2个常染色体基因座vWA、D12S391分型不符合遗传规律，所使用的Sure ID compass复合扩增系统中除1个常染色体基因座vWA分型不符合遗传规律外还有3个Y基因座DYS456、DYS458、DYS635分型不符合遗传规律。检测图谱见图4a、图4b、图5a、图5b。根据司法鉴定技术规范(SF/Z JD0105001-2010)可知，有3个以上的基因座不符合遗传规律，即可排除亲权关系的存在，故STRtyper-21G荧光检测试剂盒不能认定或排除父子关系。SureID Compass试剂盒的结果显示(见表4)被检父与被检男孩在所检测的16个常染色体基因座中只有1个基因座不符合遗传规律。被检男孩与被检父在DYS456、DYS458、DYS635这三个Y基因座上不符合遗传规律，根据司法鉴定技术规范(SF/Z JD0105001-2010)可知，有3个以上的基因座不符合遗传规律，即可以排除亲权关系的存在。因此，本发明和CN101144774相比，常染色体基因座数目已经够用，较多的Y染色体基因座则大大提高了分辨率和排除率，在单亲亲子鉴定中体现出巨大的优越性。在单亲父子关系鉴定中，需要检测常染色体基因座及Y染色体基因座，目前的做法是通过一个常染色体基因座检测试剂盒和一个Y染色体基因座检测试剂盒进行检测。在本实施例中，根据21个常染色体基因座检测结果，无法认定或排除父子关系；而增加的Y基因座最终排除了两者的父子关系。通过本发明方案，将之前所需要的两个试剂盒，即，常染色体基因座检测试剂盒和Y染色体基因座检测试剂盒整合成一个试剂盒，只需进行一次PCR扩增、电泳检测就能得到更多的信息，提高了检测效率和个体鉴别力。亲子检测试剂盒的检测结果如下表4所示。
表4：亲子检测试剂盒的检测结果
Figure PCTCN2016082019-appb-000005
上述实施例并非对本发明的限制，本发明也并不限于上述实施例。本技术领域的普通 技术人员在本发明的实质范围内，做出的变化、改型、添加或替换，也应属于本发明的保护范围。

Claims (10)

  1. 一种可同时扩增12种以上人类基因组常染色体STR基因座和12种以上Y染色体STR基因座的复合扩增方法或试剂盒。
  2. 权利要求1的复合扩增方法或试剂盒，其中将常染色体STR和Y染色体STR分别用不同的荧光进行检测。
  3. 权利要求1的复合扩增方法或试剂盒，其中所述常染色体STR基因座包含如下12种：D3S1358、D5S818、FGA、CSF1PO、D8S1179、D7S820、TH01、VWA、D13S317、D16S539、D18S51、D21S11；所述Y染色体STR基因座包含如下12种：DYS456、DYS458、DYS437、DYS439、DYS393、DYS391、DYS390、DYS635、DYS438、DYS19、GATA-H4、DYS448。
  4. 权利要求1的复合扩增方法或试剂盒，其用于案件检材分析以同时达到个体识别和家系排查(Y单倍体鉴定)的双重目的。
  5. 权利要求1的复合扩增方法或试剂盒，其用于DNA数据库建立以达到一次分析两套数据(常染色体STR和Y染色体STR)的目的；
  6. 权利要求1的复合扩增方法或试剂盒，其中利用6色荧光技术同时检测下列如下34个STR基因座：DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448、TPOX、D3S1358、D5S818、FGA、CSF1PO、D8S1179、D7S820、TH01、VWA、D13S317、D16S539、D18S51、D21S11、Y-indel、D2S1338、D19S433及Amelogenin。
  7. 权利要求6的复合扩增方法或试剂盒，其中所述34个STR基因座对应的每对引物如下：Y-indel：SEQ ID NO:1～2；D3S1358：SEQ ID NO:3～4；TH01：SEQ ID NO:5～6；D21S11：SEQ ID NO:7～8；D18S51：SEQ ID NO:9～10；D19S433：SEQ ID NO:11～12；DYS439：SEQ ID NO:13～14；DYS438：SEQ ID NO:15～16；DYS389I：SEQ ID NO:17～18；DYS448：SEQ ID NO:19～20；DYS389II：SEQ ID NO:21～22；DYS19：SEQ ID NO:23～24；GATA-H4：SEQ ID NO:25～26；DYS458：SEQ ID NO:27～28；AMEL：SEQ ID NO:29～30；D5S818：SEQ ID NO:31～32；D13S317：SEQ ID NO:33～34；D7S820：SEQ ID NO:35～36；D16S539：SEQ ID NO:37～38；CSF1PO：SEQ ID NO:39～40；D2S1338：SEQ ID NO:41～42；DYS437：SEQ ID NO:43～44；VWA：SEQ ID NO:45～46；D8S1179：SEQ ID NO:47～48；TPOX：SEQ ID NO:49～50；FGA：SEQ ID NO:51～52；DYS456：SEQ ID NO:53～54； DYS393：SEQ ID NO:55～56；DYS391：SEQ ID NO:57～58；DYS390：SEQ ID NO:59～60；DYS635：SEQ ID NO:61～62；DYS392：SEQ ID NO:63～64；DYS385a/b：SEQ ID NO:65～66。
  8. 权利要求7的复合扩增方法或试剂盒，其中每对引物中有一条引物的5’端用荧光染料标记，所选用的荧光染料为：Y-indel、D3S1358、TH01、D21S11、D18S51、D19S433、DYS439采用蓝色荧光染料FAM标记；DYS438、DYS389I、DYS448、DYS389II、DYS19、GATA-H4、DYS458采用绿色荧光染料VIC标记；AMEL、D5S818、D13S317、D7S820、D16S539、CSF1PO、D2S1338采用黄色荧光染料NED标记；DYS437、VWA、D8S1179、TPOX、FGA、DYS456采用红色荧光染料ROX标记；DYS393、DYS391、DYS390、DYS635、DYS392、DYS385a/b采用紫色荧光染料AF594。
  9. 权利要求6-8任一项的复合扩增方法或试剂盒，其中还使用或包括如下试剂：无核酸酶水、PCR缓冲液、阳性对照品、等位基因分型标准品和内标；所述无核酸酶水是无RNA酶和DNA酶的超纯水，所述阳性对照品为人类基因组DNA，所述PCR缓冲液包括DMSO 10mM、Tris-buffer 125mM、氯化钾125mM、硫酸铵65mM，所述等位基因分型标准品是试剂盒所包含的所有基因座在一定数量人群中的所有不同基因分型的混合物，所述内标是用荧光燃料标记的不同大小的片段；
  10. 权利要求6的复合扩增方法或试剂盒，其中所述引物以引物混合液形式提供，所述引物混合液包括三磷酸脱氧核苷酸(dATP、dGTP、dTTP、dCTP)7.5mM、Taq酶5U/μl、氯化镁7.5mM、BSA 2.5mg/ml、以及浓度如下的同时扩增34个基因座的所有引物：SEQ ID NO:1～2：0.84μM；SEQ ID NO:3～4：0.5μM；SEQ ID NO:5～6：0.67μM；SEQ ID NO:7～8：0.66μM；SEQ ID NO:9～10：0.67μM；SEQ ID NO:11～12：1.34μM；SEQ ID NO:13～14：0.54μM；SEQ ID NO:15～16：1.26μM；SEQ ID NO:17～18：0.3μM；SEQ ID NO:19～20：0.7μM；SEQ ID NO:21～22：1.36μM；SEQ ID NO:23～24：0.65μM；SEQ ID NO:25～26：0.5μM；SEQ ID NO:27～28：0.6μM；SEQ ID NO:29～30：0.65μM；SEQ ID NO:31～32：1.3μM；SEQ ID NO:33～34：1.2μM；SEQ ID NO:35～36：1.3μM；SEQ ID NO:37～38：1.5μM；SEQ ID NO:39～40：4.6μM；SEQ ID NO:41～42：1.8μM；SEQ ID NO:43～44：2.7μM；SEQ ID NO:45～46：1.9μM；SEQ ID NO:47～48：2.0μM；SEQ ID NO:49～50：2.1μM；SEQ ID NO:51：2.0μM；SEQ ID NO:52：2.1μM；SEQ ID NO:53：2.2μM；SEQ ID NO:54：2.1μM；SEQ ID NO:55：2.5μM；SEQ ID NO:56：2.3μM；SEQ ID NO:57：2.6μM；SEQ ID NO:58：2.3μM；SEQ ID NO:59：2.7μM；SEQ ID  NO: 60：2.4 μM；SEQ ID NO: 61：2.5 μM；SEQ ID NO: 62：2.3 μM；SEQ ID NO: 63：3.1μM； SEQ ID NO: 64： 3.0 μM； SEQ ID NO: 65： 2.7 μM； SEQ ID NO: 66： 2.9 μM。
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