WO2016066070A1 - Y chromosome str locus fluorescent labeling multiplex amplification kit having enhanced identification capability and use thereof - Google Patents
Y chromosome str locus fluorescent labeling multiplex amplification kit having enhanced identification capability and use thereof Download PDFInfo
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- the invention relates to a PCR amplification kit, in particular to a fluorescent composite amplification kit for simultaneously detecting 27 Y chromosome loci in a single tube, and the invention also relates to a preparation method of the fluorescent composite amplification kit and the reagent
- the application of the box in the field of forensic identification belongs to the field of Y chromosome typing and identification.
- 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, which was developed by ReliaGene Technologies in 2001. It can amplify DYS19, DYS389II, DYS390, DYS391, DYS393, DYS385a/b.
- ReliaGene Technologies developed a compound amplification.
- 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.
- the Y chromosome genetic marker is located in the non-recombination region of the Y chromosome, and the entire non-recombination region is equivalent to a genetic marker. Therefore, the personal recognition ability and the paternity test ability of the Y chromosome genetic marker cannot use the multiplication principle like the autosome. Therefore, in order to achieve sufficient exclusion probability and personal recognition probability, it is necessary to continuously increase the new Y chromosome genetic marker. Therefore, in order to improve the individual identification ability, the Y-STR kit needs to add a plurality of new Y chromosome genetic markers with high genetic polymorphism on the existing basis.
- a Y-chromosome STR locus fluorescent labeling complex amplification kit with enhanced discrimination capability which mainly comprises three types of loci, wherein 20 loci mutation rates are less than 1 ⁇ for conventional paternal genetic testing; In addition, 7 loci with a mutation rate greater than 1% have been added, which are mainly used to increase the recognition of close relatives. The resolution is ahead of similar products in the domestic market; in addition, 8 amplified fragments are included in the kit.
- the present invention aims at the above technical problems, and deeply studies the genetic polymorphism of the Y-STR locus in the male population, and provides a fluorescent marker complex amplification kit for the Y chromosome STR locus with enhanced discrimination ability. Forensic identification and paternity testing.
- the present invention passes the DYS456, DY508, A10, DYS463, DYS510, DYS464, DYS458, DYS520, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS447, DYS438, DYS389I, DYS19, GATA-H4, Genetic polymorphisms of 37 loci in DYS389II, DYS448, DYS576, DYS570, DYS481, DYF387S1, DYS627, DYS460, DYS449, DYS533, DYS518, etc.
- DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 27 loci including DYS458, DYS460, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS449, DYS533, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448 and DYS518.
- These include 17 loci that can be amplified by the Yfiler kit launched by AB, which is currently used in China.
- Y-STR loci including 9 European minimum haplotypes and 2 SWGDAM recommended loci, and on this basis, they have been added.
- Ten highly polymorphic Y-STR loci including DYS576, DYS570, DYS481, DYS627, DYS460, DYS449, DYS533, DYS518, DYF387S1 (including two loci, DYF387S1a and DYF387S1b). These loci are compatible with existing kits, ensuring the sharing and communication of existing DNA data, and can improve the cumulative individual recognition ability and cumulative non-parent exclusion rate of DNA testing systems, and are more in line with the technical requirements of DNA testing.
- the newly added DYS576, DYS570, DYS627, DYS449, DYS518, and DYF387S1 mutations with more than 1% mutation rate can increase the recognition of close relatives. This is the first time in domestic kits.
- the present invention provides a Y-chromosome STR locus fluorescent labeling complex amplification kit with enhanced discrimination ability, comprising the following 17 Y chromosome STR loci specificity Oligonucleotide amplification primer pairs: DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448; Specific oligonucleotide amplification of a Y chromosome STR locus Primer pairs: at least one of DYS576, DYS570, DYS481, DYS627, DYS460, DYS449, DYS533, DYS518, and DYF387S1
- the kit provided by the present invention comprises the following specific oligonucleotide amplification primers for 27 Y chromosome STR loci: DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390 , DYS635, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448, DYS576, DYS570, DYS481, DYS627, DYS460, DYS449, DYS533, DYS518 and DYF387S1.
- Table 1 The specific information of the 27 Y-STR loci is shown in Table 1.
- each pair of primers has a certain primer sequence for amplifying the corresponding Y-STR locus, and the corresponding relationship is shown in Table 2:
- the concentration of the primer corresponding to each locus in the kit of the present invention is as shown in Table 2, and thus there is no non-specific peak in the results thus measured.
- 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: DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 adopt FAM label; DYS458, DYS460, DYS437, DYS439 , DYS392, DYS385a/b adopt HEX mark; DYS393, DYS391, DYS390, DYS635, DYS449, DYS533 adopt TAMRA mark; DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448 and DYS518 adopt ROX mark; internal standard adopts orange fluorescent mark
- the fluorescent label is Atto 633.
- 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 invention provides a Y chromosome STR locus fluorescent labeling composite amplification kit with enhanced discrimination ability. These include nuclease-free water, 2 ⁇ PCR Master Mix, STRtyper-27Y Primer Mix, positive control, Allelic ladder allelic ladder, Size-500 orange fluorescent molecular weight internal standard, and spectral calibration standards. It is worth mentioning that the 2 ⁇ 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.
- Bokun saliva card, hair, oral exfoliated cells, DNA extraction and other samples this has not been done in domestic or even foreign kits, in addition, this improved buffer can greatly improve
- the amplification efficiency 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 similar products on the market at home and abroad, thereby greatly increasing the cumulative individual recognition power and cumulative non-parent exclusion rate of the system, and generally improving the individual. Discrimination.
- the present invention also innovatively includes seven rapid mutation loci: DYS576, DYS570, DYS627, DYS449, DYS518, DYF387S1 (including DYF387S1a and DYF387S1b), which further improves the recognition of close relatives, making the kit practical and functional.
- Great increase get rid of the strange circle of domestic kit developers to increase the same type of detection locus.
- Figure 1 shows the allelic ladder of the fluorescent-labeled complex amplification assay system for the 27 loci of the Y chromosome.
- Figure 2 is a Y-STR sub-graph of the male standard 007.
- Figure 3 is a Y-STR subgraph of the scorpion.
- Figure 4 is a Y-STR sub-graph of the uncle.
- the present invention passes the DYS456, DY508, A10, DYS463, DYS510, DYS464, DYS458, DYS520, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS447, DYS438, DYS389I, DYS19, GATA-H4, Genetic polymorphisms of 37 loci in DYS389II, DYS448, DYS576, DYS570, DYS481, DYF387S1, DYS627, DYS460, DYS449, DYS533, DYS518, etc.
- loci are compatible with existing kits, ensuring the sharing and communication of existing DNA data, and can improve the cumulative individual recognition ability and cumulative non-parent exclusion rate of DNA testing systems, and are more in line with the technical requirements of DNA testing.
- the 7 rapid mutation loci DYS576, DYS570, DYS627, DYS449, DYS518, DYF387S1 (including DYF387S1a and DYF387S1b) with a mutation rate greater than 1% can increase the recognition of close relatives in an unprecedented manner. It is the first time.
- the invention has identified and selected fluorescent dyes, and selected five kinds of fluorescent labels of blue, green, yellow, red and orange to construct a 5-color fluorescent combination scheme. Based on the determination of the 5-color fluorescence combination scheme, the gene locus combination method and the fluorescent label type were designed through a large number of repeated experiments. Considering the production cost and the amplification efficiency of each locus primer, 27 loci were divided into 4 groups, using FAM, HEX, TAMRA, ROX group labeling, and the molecular weight internal standard was carried out with the fifth color orange fluorescent dye Atto 633. mark.
- DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 are labeled with FAM;
- DYS458, DYS460, DYS437, DYS439, DYS392, DYS385a/b are labeled with HEX;
- DYS393, DYS391 DYSRA, DYS635, DYS449, and DYS533 were labeled with TAMRA;
- DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448, and DYS518 were labeled with ROX; the internal standard was selected with orange fluorescent label and the fluorescent label was Atto 633.
- This combination of loci allows simultaneous detection and analysis of 27 loci with only five types of fluorescence.
- the PCR Master Mix includes: DMSO 10 mM, Tris-buffer 125 mM, potassium chloride 125 mM, sulfur Ammonium phosphate 65 mM can be used to achieve a wide range of common materials in the market.
- the STRtyper-27Y Primer Mix includes all primers for amplifying 27 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 2.5 mg/ml.
- Control DNA 9948A is human genomic DNA purchased from Suzhou Xinhai Biotechnology Co., Ltd.
- the Allelic Ladder allelic typing standard is a collection of distributions of alleles of all different genotypes of the locus contained in the kit in a certain number of populations.
- the Size-500 orange fluorescent molecular weight internal standard is a series of plasmids used to calibrate a certain fragment size from Ningbo Haiershi Gene Technology Co., Ltd.
- the spectral calibration standard is a fluorescent PCR amplification product of five 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 consisting of deionized formamide and a molecular weight internal standard (Size-500) in the system ⁇ (1 ⁇ L Size-500+12) ⁇ L deionized formamide) ⁇ (injection number) ⁇ .
- 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 27 loci in the present invention tests pigs, dogs, sheep, ducks, chickens, mice, cows, E. coli, etc., without specific amplification peaks, indicating that the system has Species specificity.
- the kit provided by the present invention is used for the paternity test of the tert- ⁇ relationship, and the measurement steps are as follows:
- the paternity test sample is provided by a judicial identification office.
- 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 detection template.
- Amplification detection According to Examples 2 to 4, fluorescent labeling, PCR amplification and genetic analysis were carried out, and a kit for amplifying primer pairs of a specific oligonucleotide containing 27 Y chromosome STR loci was used.
- the classification results of the scorpion are shown in Figure 3.
- the results of the uncle's classification are shown in Figure 4.
- the comparison results are shown in the following table:
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Abstract
Disclosed is a Y chromosome STR locus fluorescent labeling multiplex amplification kit having an enhanced identification capability, and using same to detect DNA genes can amplify and analyze the following 27 STR loci: DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448, DYS576, DYS570, DYS481, DYF387S1, DYS627, DYS460, DYS449, DYS533 and DYS518.
Description
本发明涉及一种PCR扩增试剂盒，尤其涉及一种单管同时检测27个Y染色体基因座的荧光复合扩增试剂盒，本发明还涉及该荧光复合扩增试剂盒的制备方法以及该试剂盒在司法鉴定领域中的应用，属于Y染色体分型和鉴定领域。The invention relates to a PCR amplification kit, in particular to a fluorescent composite amplification kit for simultaneously detecting 27 Y chromosome loci in a single tube, and the invention also relates to a preparation method of the fluorescent composite amplification kit and the reagent The application of the box in the field of forensic identification belongs to the field of Y chromosome typing and identification.
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。A short tandem repeat (STR) 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. At the same time, 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.
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，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。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. . Due to these characteristics of the Y chromosome, 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. 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.
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。In the field of forensic identification and paternity testing, DNA analysis relies mainly on commercial kits. The first Y-STR kit is Y-PLEXTM6, which was developed by ReliaGene Technologies in 2001. It can amplify DYS19, DYS389II, DYS390, DYS391, DYS393, DYS385a/b. In 2002, ReliaGene Technologies developed a compound amplification. Y-PLEXTM5 of DYS389I/II, DYS439, DYS438, DYS392, to 2003
In September, ReliaGene Technologies introduced Y-PLEXTM12, which integrates all Y-PLEXTM6 and Y-PLEXTM5 loci. In October of the same year, Promega introduced the PowerPlex Y kit, which can amplify 12 loci, including DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439. Applied Biosystems (abbreviated AB) has released a yfilerTM kit containing 17 Y-STR loci, which adds another five highly polymorphic Y-STR loci to PowerPlex Y, including DYS456. , DYS458, DYS635, GATA-H4, DYS448. In the fall of 2012, Promega introduced the PowerPlex Y23 kit, and added 6 new loci in addition to the 17 loci compatible with the yfilerTM kit, including DYS549, DYS481, and DYS533. , DYS570, DYS576, DYS643, further improve the resolution of the kit. At present, 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. At present, there are mainly Goldeneye 20Y kits from Beijing Basic Point Cognitive Company, which can amplify 20 Y-STR loci in addition to 17 loci including yfilerTM kit. Added DYS460, DYS447, DYS388. In addition, 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.
然而长期的DNA检验试验表明，STR基因座的遗传多态性在种族间存在一定差异，各个基因座之间的差别也很大。由此给DNA检验技术的应用及其效率造成一定影响。Y染色体遗传标记位于Y染色体的非重组区，整个非重组区相当于一个遗传标记，因此Y染色体遗传标记的个人识别能力和亲权鉴定能力不能像常染色体那样使用相乘原则。所以，为了达到足够的排除概率和个人识别概率，就必须不断的增加新的Y染色体遗传标记。所以，为了提高个人鉴别能力，Y-STR试剂盒就需要在现有基础上增加多个具有高度遗传多态性的新的Y染色体遗传标记。However, long-term DNA testing experiments show that the genetic polymorphism of the STR locus varies from race to race, and the differences between the loci are also large. This has a certain impact on the application of DNA testing technology and its efficiency. The Y chromosome genetic marker is located in the non-recombination region of the Y chromosome, and the entire non-recombination region is equivalent to a genetic marker. Therefore, the personal recognition ability and the paternity test ability of the Y chromosome genetic marker cannot use the multiplication principle like the autosome. Therefore, in order to achieve sufficient exclusion probability and personal recognition probability, it is necessary to continuously increase the new Y chromosome genetic marker. Therefore, in order to improve the individual identification ability, the Y-STR kit needs to add a plurality of new Y chromosome genetic markers with high genetic polymorphism on the existing basis.
由此，为了进一步改善市售扩增试剂盒的灵敏性和效率及识别度，申请人对男性人群的Y-STR基因座的遗传多态性进行了深入的研究，并以此为依据开发了一种具有增强鉴别能力的Y染色体STR基因座荧光标记复合扩增试剂盒，其主要包括了三种类型的基因座，其中20个基因座突变率小于1‰，用于常规的父系遗传检测；另外增加了7个突变率大于1％的基因座，主要用于增加近亲男性的识别度，其分辨率领先于国内市场上的同类产品；此外，在本试剂盒中包含了8个扩增片段长度小于220bp的基因座，即miniSTR。MiniSTR主要用于降解检材检测实验，数量上领先于市场上的同类产品，识别度也高于
市场上的同类产品。此种多用途试剂盒目前尚未见报道与使用。Therefore, in order to further improve the sensitivity, efficiency and recognition of commercially available amplification kits, the applicant has conducted in-depth research on the genetic polymorphism of the Y-STR locus in the male population and developed it based on this. A Y-chromosome STR locus fluorescent labeling complex amplification kit with enhanced discrimination capability, which mainly comprises three types of loci, wherein 20 loci mutation rates are less than 1‰ for conventional paternal genetic testing; In addition, 7 loci with a mutation rate greater than 1% have been added, which are mainly used to increase the recognition of close relatives. The resolution is ahead of similar products in the domestic market; in addition, 8 amplified fragments are included in the kit. A locus less than 220 bp in length, ie miniSTR. MiniSTR is mainly used for degradable material testing experiments. It is ahead of similar products in the market in quantity, and its recognition is higher than that.
Similar products on the market. Such multi-purpose kits have not been reported and used at present.
发明内容Summary of the invention
本发明针对以上技术问题，对男性人群的Y-STR基因座的遗传多态性进行了深入的研究，提供了一种具有增强鉴别能力的Y染色体STR基因座荧光标记复合扩增试剂盒，用于法医鉴定及亲子鉴定。The present invention aims at the above technical problems, and deeply studies the genetic polymorphism of the Y-STR locus in the male population, and provides a fluorescent marker complex amplification kit for the Y chromosome STR locus with enhanced discrimination ability. Forensic identification and paternity testing.
本发明所采用的技术方案为：The technical solution adopted by the invention is:
(1)选择具有增强鉴别能力的Y染色体STR基因座。本发明通过对DYS456、DY508、A10、DYS463、DYS510、DYS464、DYS458、DYS520、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS447、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448、DYS576、DYS570、DYS481、DYF387S1、DYS627、DYS460、DYS449、DYS533、DYS518等37个基因座在男性人群中的遗传多态性调查，最后确定DYS456、DYS576、DYS570、DYS481、DYF387S1、DYS627、DYS458、DYS460、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS449、DYS533、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448和DYS518等27个基因座。其中包括目前国内使用的AB公司推出的Yfiler试剂盒可扩增的17个基因座，这其中已包括9个欧洲最小单倍型和2个SWGDAM推荐基因座，并在此基础上，又增加了10个具有高度多态性的Y-STR基因座，包括DYS576、DYS570、DYS481、DYS627、DYS460、DYS449、DYS533、DYS518、DYF387S1(包括两个基因座，即DYF387S1a和DYF387S1b)。这些基因座既与现有试剂盒兼容，保证已有DNA数据的共享与交流，又能够提高DNA检验***的累积个体识别能力和累积非父排除率，更加符合DNA检验的技术要求。特别是新增的DYS576、DYS570、DYS627、DYS449、DYS518、DYF387S1突变率大于1％的7个快速突变基因座，可以空前地提高近亲男性的识别度，这在国内试剂盒尚属首次。(1) Select a Y chromosome STR locus with enhanced discrimination ability. The present invention passes the DYS456, DY508, A10, DYS463, DYS510, DYS464, DYS458, DYS520, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS447, DYS438, DYS389I, DYS19, GATA-H4, Genetic polymorphisms of 37 loci in DYS389II, DYS448, DYS576, DYS570, DYS481, DYF387S1, DYS627, DYS460, DYS449, DYS533, DYS518, etc. in male population, and finally determined DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 27 loci, including DYS458, DYS460, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS449, DYS533, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448 and DYS518. These include 17 loci that can be amplified by the Yfiler kit launched by AB, which is currently used in China. These include 9 European minimum haplotypes and 2 SWGDAM recommended loci, and on this basis, they have been added. Ten highly polymorphic Y-STR loci, including DYS576, DYS570, DYS481, DYS627, DYS460, DYS449, DYS533, DYS518, DYF387S1 (including two loci, DYF387S1a and DYF387S1b). These loci are compatible with existing kits, ensuring the sharing and communication of existing DNA data, and can improve the cumulative individual recognition ability and cumulative non-parent exclusion rate of DNA testing systems, and are more in line with the technical requirements of DNA testing. In particular, the newly added DYS576, DYS570, DYS627, DYS449, DYS518, and DYF387S1 mutations with more than 1% mutation rate can increase the recognition of close relatives. This is the first time in domestic kits.
(2)确定具有增强鉴别能力的Y-STR荧光标记复合扩增试剂盒。(2) Determining a Y-STR fluorescent labeled composite amplification kit with enhanced discrimination ability.
基于以上具有增强鉴别能力的Y-STR基因座的确定，本发明提供一种具有增强鉴别能力的Y染色体STR基因座荧光标记复合扩增试剂盒，包含下列17个Y染色体STR基因座的特异性寡核苷酸扩增引物对：DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448；同时还包含下列10个Y染色体STR基因座的特异性寡核苷酸扩增
引物对：DYS576、DYS570、DYS481、DYS627、DYS460、DYS449、DYS533、DYS518和DYF387S1(包括DYF387S1a和DYF387S1b)中的至少一个。Based on the above determination of the Y-STR locus with enhanced discriminating ability, the present invention provides a Y-chromosome STR locus fluorescent labeling complex amplification kit with enhanced discrimination ability, comprising the following 17 Y chromosome STR loci specificity Oligonucleotide amplification primer pairs: DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448; Specific oligonucleotide amplification of a Y chromosome STR locus
Primer pairs: at least one of DYS576, DYS570, DYS481, DYS627, DYS460, DYS449, DYS533, DYS518, and DYF387S1 (including DYF387S1a and DYF387S1b).
优选地，本发明所提供的试剂盒同时包含下列27个Y染色体STR基因座的特异性寡核苷酸扩增引物：DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448、DYS576、DYS570、DYS481、DYS627、DYS460、DYS449、DYS533、DYS518和DYF387S1。所述27个Y-STR基因座的具体信息见表1。Preferably, the kit provided by the present invention comprises the following specific oligonucleotide amplification primers for 27 Y chromosome STR loci: DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390 , DYS635, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448, DYS576, DYS570, DYS481, DYS627, DYS460, DYS449, DYS533, DYS518 and DYF387S1. The specific information of the 27 Y-STR loci is shown in Table 1.
表1：27个Y-STR基因座的相关信息Table 1: Information on 27 Y-STR loci
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Y-STR基因座，其对应关系如表2所示：Further, each pair of primers has a certain primer sequence for amplifying the corresponding Y-STR locus, and the corresponding relationship is shown in Table 2:
表2：各基因座对应的引物序列及其浓度Table 2: Primer sequences and their concentrations for each locus
优选地，本发明的试剂盒中所述各基因座对应的引物的浓度如表2所示，这样测出的结果中无非特异性峰。Preferably, the concentration of the primer corresponding to each locus in the kit of the present invention is as shown in Table 2, and thus there is no non-specific peak in the results thus measured.
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。Further, 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.
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：DYS456、DYS576、DYS570、DYS481、DYF387S1、DYS627采用FAM标记；DYS458、DYS460、DYS437、DYS439、DYS392、DYS385a/b采用HEX标记；DYS393、DYS391、DYS390、DYS635、DYS449、DYS533采用TAMRA标记；DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448和DYS518采用ROX标记；内标选用橙色荧光标记，荧光标记物为Atto 633。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: DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 adopt FAM label; DYS458, DYS460, DYS437, DYS439 , DYS392, DYS385a/b adopt HEX mark; DYS393, DYS391, DYS390, DYS635, DYS449, DYS533 adopt TAMRA mark; DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448 and DYS518 adopt ROX mark; internal standard adopts orange fluorescent mark The fluorescent label is 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、***、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。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.
本发明提供的具有增强鉴别能力的Y染色体STR基因座荧光标记复合扩增试剂盒还
包括无核酸酶水、2×PCR Master Mix、STRtyper-27Y Primer Mix、阳性对照品、Allelic ladder等位基因分型标准品、Size-500橙色荧光分子量内标以及光谱校正标准物。值得一提的是本发明所采用的2×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的提高扩增效率，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。The invention provides a Y chromosome STR locus fluorescent labeling composite amplification kit with enhanced discrimination ability.
These include nuclease-free water, 2×PCR Master Mix, STRtyper-27Y Primer Mix, positive control, Allelic ladder allelic ladder, Size-500 orange fluorescent molecular weight internal standard, and spectral calibration standards. It is worth mentioning that the 2×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. , Bokun saliva card, hair, oral exfoliated cells, DNA extraction and other samples, this has not been done in domestic or even foreign kits, in addition, this improved buffer can greatly improve The amplification efficiency 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.
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的同类产品，从而极大地提高了***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。同时本发明还创新性地包括7个快速突变基因座：DYS576、DYS570、DYS627、DYS449、DYS518、DYF387S1(包括DYF387S1a和DYF387S1b)，进一步提高了近亲男性的识别度，使得试剂盒实用性和功能性大增，摆脱了国内试剂盒开发厂商一味增加同类检测基因座的怪圈。In summary, the kits provided by the present invention detect more gene loci than the similar products on the market at home and abroad, thereby greatly increasing the cumulative individual recognition power and cumulative non-parent exclusion rate of the system, and generally improving the individual. Discrimination. At the same time, the present invention also innovatively includes seven rapid mutation loci: DYS576, DYS570, DYS627, DYS449, DYS518, DYF387S1 (including DYF387S1a and DYF387S1b), which further improves the recognition of close relatives, making the kit practical and functional. Great increase, get rid of the strange circle of domestic kit developers to increase the same type of detection locus.
图1为Y染色体27个基因座的荧光标记复合扩增检验***等位基因分型标准物。Figure 1 shows the allelic ladder of the fluorescent-labeled complex amplification assay system for the 27 loci of the Y chromosome.
图2为男性标准品007的Y-STR分型图。Figure 2 is a Y-STR sub-graph of the male standard 007.
图3为侄子的Y-STR分型图。Figure 3 is a Y-STR subgraph of the scorpion.
图4为叔叔的Y-STR分型图。Figure 4 is a Y-STR sub-graph of the uncle.
以下结合附图对本发明作进一步详细描述。The invention is further described in detail below with reference to the accompanying drawings.
实施例1基因座的确定Determination of the locus of Example 1
本发明通过对DYS456、DY508、A10、DYS463、DYS510、DYS464、DYS458、DYS520、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS447、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448、DYS576、DYS570、DYS481、DYF387S1、DYS627、DYS460、DYS449、DYS533、DYS518等37个基因座在男性人群中的遗传多态性调查，最后确定DYS456、DYS576、DYS570、DYS481、DYF387S1、DYS627、DYS458、DYS460、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、
DYS390、DYS635、DYS449、DYS533、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448和DYS518等27个基因座。这些基因座既与现有试剂盒兼容，保证已有DNA数据的共享与交流，又能够提高DNA检验***的累积个体识别能力和累积非父排除率，更加符合DNA检验的技术要求。特别是新增的突变率大于1％的7个快速突变基因座DYS576、DYS570、DYS627、DYS449、DYS518、DYF387S1(包括DYF387S1a和DYF387S1b)，可以空前地提高近亲男性的识别度，这在国内试剂盒尚属首次。The present invention passes the DYS456, DY508, A10, DYS463, DYS510, DYS464, DYS458, DYS520, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS447, DYS438, DYS389I, DYS19, GATA-H4, Genetic polymorphisms of 37 loci in DYS389II, DYS448, DYS576, DYS570, DYS481, DYF387S1, DYS627, DYS460, DYS449, DYS533, DYS518, etc. in male population, and finally determined DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 , DYS458, DYS460, DYS437, DYS439, DYS392, DYS385a/b, DYS393, DYS391,
27 loci including DYS390, DYS635, DYS449, DYS533, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448 and DYS518. These loci are compatible with existing kits, ensuring the sharing and communication of existing DNA data, and can improve the cumulative individual recognition ability and cumulative non-parent exclusion rate of DNA testing systems, and are more in line with the technical requirements of DNA testing. In particular, the 7 rapid mutation loci DYS576, DYS570, DYS627, DYS449, DYS518, DYF387S1 (including DYF387S1a and DYF387S1b) with a mutation rate greater than 1% can increase the recognition of close relatives in an unprecedented manner. It is the first time.
实施例2荧光标记复合扩增体系的基因座组合方案设计Example 2 Design of gene locus combination scheme for fluorescent labeling complex amplification system
本发明对荧光染料进行了鉴别、遴选，选用了蓝、绿、黄、红、橙五种荧光标记物，构建了5色荧光组合方案。在确定5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27个基因座分成4组，使用FAM、HEX、TAMRA、ROX分组标记，分子量内标用第5种颜色橙色的荧光染料Atto 633进行标记。经过筛选最终确定一种优选的荧光染料标记的方法为：DYS456、DYS576、DYS570、DYS481、DYF387S1、DYS627采用FAM标记；DYS458、DYS460、DYS437、DYS439、DYS392、DYS385a/b采用HEX标记；DYS393、DYS391、DYS390、DYS635、DYS449、DYS533采用TAMRA标记；DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448和DYS518采用ROX标记；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5种荧光就可以实现27个基因座同时检测分析。The invention has identified and selected fluorescent dyes, and selected five kinds of fluorescent labels of blue, green, yellow, red and orange to construct a 5-color fluorescent combination scheme. Based on the determination of the 5-color fluorescence combination scheme, the gene locus combination method and the fluorescent label type were designed through a large number of repeated experiments. Considering the production cost and the amplification efficiency of each locus primer, 27 loci were divided into 4 groups, using FAM, HEX, TAMRA, ROX group labeling, and the molecular weight internal standard was carried out with the fifth color orange fluorescent dye Atto 633. mark. After screening, a preferred method for identifying fluorescent dyes is as follows: DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 are labeled with FAM; DYS458, DYS460, DYS437, DYS439, DYS392, DYS385a/b are labeled with HEX; DYS393, DYS391 DYSRA, DYS635, DYS449, and DYS533 were labeled with TAMRA; DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448, and DYS518 were labeled with ROX; the internal standard was selected with orange fluorescent label and the fluorescent label was Atto 633. This combination of loci allows simultaneous detection and analysis of 27 loci with only five types of fluorescence.
实施例3扩增基因座及其产物检测的实验过程Example 3 Experimental procedure for amplification of loci and detection of their products
本发明提供的具有增强鉴别能力的Y染色体STR基因座荧光标记复合扩增试剂盒包括：The Y-chromosome STR locus fluorescent labeling composite amplification kit with enhanced discrimination ability provided by the invention comprises:
1)2×PCR Master Mix1) 2×PCR Master Mix
2)STRtyper-27Y Primer Mix2) STRtyper-27Y Primer Mix
3)Control DNA 9948A3) Control DNA 9948A
4)Allelic Ladder等位基因分型标准品4) Allelic Ladder allelic standard
5)Size-500橙色荧光分子量内标5) Size-500 orange fluorescent molecular weight internal standard
6)光谱校正标准物6) Spectral calibration standards
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。The PCR Master Mix includes: DMSO 10 mM, Tris-buffer 125 mM, potassium chloride 125 mM, sulfur
Ammonium phosphate 65 mM can be used to achieve a wide range of common materials in the market.
所述STRtyper-27Y Primer Mix包括扩增27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、BSA2.5mg/ml。The STRtyper-27Y Primer Mix includes all primers for amplifying 27 loci (see Table 2 for concentration), deoxynucleotide triphosphate (dATP, dGTP, dTTP, dCTP) 7.5 mM, Taq 5 U/μl, magnesium chloride 7.5 mM, BSA 2.5 mg/ml.
所述Control DNA 9948A是人类基因组DNA，购自苏州新海生物科技有限公司。The Control DNA 9948A is human genomic DNA purchased from Suzhou Xinhai Biotechnology Co., Ltd.
所述Allelic Ladder等位基因分型标准品是试剂盒所包含基因座所有不同基因分型的各等位基因在一定数量人群中的分布情况集合。The Allelic Ladder allelic typing standard is a collection of distributions of alleles of all different genotypes of the locus contained in the kit in a certain number of populations.
所述Size-500橙色荧光分子量内标是一系列用于标定一定片段大小的质粒，来自宁波海尔施基因科技有限公司。The Size-500 orange fluorescent molecular weight internal standard is a series of plasmids used to calibrate a certain fragment size from Ningbo Haiershi Gene Technology Co., Ltd.
所述光谱校正标准物是5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。The spectral calibration standard is a fluorescent PCR amplification product of five different size fragments from Ningbo Haiershi Gene Technology Co., Ltd.
1、反应体系的配置1. Configuration of the reaction system
| 组分Component | 体积volume |
| 无核酸酶水(无RNA酶/DNA酶超纯水Dnase/Rnase Free)Nuclease-free water (no RNase/DNase ultrapure water Dnase/Rnase Free) |
补足至25.0μLMake up to 25.0 |
| 2×PCR Master Mix2×PCR Master Mix | 12.5μL12.5 μL |
| STRtyper-27Y Primer MixSTRtyper-27Y Primer Mix | 6.25μL6.25μL |
| Control DNA 9948AControl DNA 9948A | 1ng1ng |
| 总total | 25μL25μL |
2、扩增热循环实验方案2, amplification thermal cycle experimental program
1)将PCR扩增管置于热循环仪上；1) placing the PCR amplification tube on a thermal cycler;
2)选择表3所示的程序进行扩增；2) Select the program shown in Table 3 for amplification;
3)扩增后的样品应避光保存；3) The amplified sample should be stored in the dark;
表3：热循环仪的扩增程序Table 3: Amplification procedure for thermal cycler
|
步骤 | 温度temperature | 时间time | |
| 11 | 95℃95 ° C |
5分钟5 |
|
| 22 |
94℃94° |
10秒钟10 seconds | |
| 33 |
61℃61 ° |
1分钟1 |
|
| 44 |
70℃70 ° |
30秒钟30 seconds | |
| 55 | N/AN/A |
重复2-4步骤27次(共28次)Repeat 2-4 |
|
| 66 |
60℃60 ° |
20分钟20 |
|
| 77 | 4℃4 ° C | 持续:直至收取PCR产物Continuous: until the PCR product is collected |
3、扩增产物在遗传分析仪上荧光检测3. Fluorescence detection of amplification products on a genetic analyzer
由去离子甲酰胺与***中分子量内标(Size-500)组成上样混合物{(1μL Size-500+12
μL去离子甲酰胺)×(进样数)}。将9μL上样混合物与1μL扩增产物或***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。A sample mixture consisting of deionized formamide and a molecular weight internal standard (Size-500) in the system {(1 μL Size-500+12)
μL deionized formamide) × (injection number)}. Mix 9 μL of the sample mixture with 1 μL of amplification product or System Allelic ladder to avoid air bubbles and electrophoresis as soon as possible. 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.
实施例4检测试剂盒的灵敏度、特异性分析Example 4 Sensitivity and specificity analysis of the test kit
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。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.
特异性分析：本发明中27个基因座的荧光标记复合扩增检验***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***具有种属特异性。Specificity analysis: The fluorescent labeling complex amplification test system of 27 loci in the present invention tests pigs, dogs, sheep, ducks, chickens, mice, cows, E. coli, etc., without specific amplification peaks, indicating that the system has Species specificity.
以上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。The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments. Variations, modifications, additions or substitutions made by those skilled in the art within the scope of the invention are also within the scope of the invention.
实施例5本发明所提供的试剂盒在亲子鉴定中的应用Example 5 Application of the kit provided by the present invention in paternity testing
将本发明所提供的试剂盒用于叔-侄关系的亲子鉴定，测定步骤如下：The kit provided by the present invention is used for the paternity test of the tert-侄 relationship, and the measurement steps are as follows:
1.收集亲子鉴定案件中的血斑：亲子鉴定样本由某司法鉴定所提供。1. Collect blood spots in paternity test cases: The paternity test sample is provided by a judicial identification office.
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。2. Treatment of the sample to be inspected: 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 detection template.
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27个Y染色体STR基因座的特异性寡核苷酸扩增引物对的试剂盒，侄子的分型结果见图3，叔叔的分型结果见图4，其对比结果见下表：3. Amplification detection: According to Examples 2 to 4, fluorescent labeling, PCR amplification and genetic analysis were carried out, and a kit for amplifying primer pairs of a specific oligonucleotide containing 27 Y chromosome STR loci was used. The classification results of the scorpion are shown in Figure 3. The results of the uncle's classification are shown in Figure 4. The comparison results are shown in the following table:
结果显示：叔-侄27个Y-STR的检测结果完全一致，说明他们属于同一个男性家族。但是Y染色体检测的特殊性，并不能确定他们间叔侄关系，需要进一步对常染色体，甚至更多家庭成员进行检测。所以，本发明提供的试剂盒在亲子鉴定中一般作为常染色体鉴定结果的补充与佐证更为复杂。在实施例中所涉及的工艺条件在有效窗口内可以进行合理改变且达到本发明所揭示的效果。
The results showed that the results of the 27-Y-STR of the uncle-侄 were identical, indicating that they belonged to the same male family. However, the particularity of the Y chromosome detection does not determine the relationship between them. It is necessary to further detect autosomes and even more family members. Therefore, the kit provided by the present invention is more complicated and supplemented as a supplement to autosomal identification results in paternity testing. The process conditions involved in the examples can be reasonably changed within the effective window and achieve the effects disclosed herein.
Claims (9)
- 一种Y染色体STR基因座荧光标记复合扩增试剂盒，其包含下列Y染色体STR基因座的特异性寡核苷酸扩增引物对：DYS456、DYS458、DYS437、DYS439、DYS392、DYS385a/b、DYS393、DYS391、DYS390、DYS635、DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448、DYS576、DYS570、DYS481、DYS627、DYS460、DYS449、DYS533、DYS518、DYF387S11。A Y-chromosome STR locus fluorescent labeling complex amplification kit comprising the following specific oligonucleotide amplification primer pairs for the Y chromosome STR locus: DYS456, DYS458, DYS437, DYS439, DYS392, DYS385a/b, DYS393 , DYS391, DYS390, DYS635, DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448, DYS576, DYS570, DYS481, DYS627, DYS460, DYS449, DYS533, DYS518, DYF387S11.
- 权利要求1的试剂盒，其中所述各STR基因座对应的引物对序列如下：DYS456：SEQ ID NO:1～2；DYS458：SEQ ID NO:3～4；DYS437：SEQ ID NO:5～6；DYS439：SEQ ID NO:7～8；DYS392：SEQ ID NO:9～10；DYS385a/b：SEQ ID NO:11～12；DYS393：SEQ ID NO:13～14；DYS391：SEQ ID NO:15～16；DYS390：SEQ ID NO:17～18；DYS635：SEQ ID NO:19～20；DYS438：SEQ ID NO:21～22；DYS389I：SEQ ID NO:23～24；DYS19：SEQ ID NO:25～26；GATA-H4：SEQ ID NO:27～28；DYS389II：SEQ ID NO:29～30；DYS448：SEQ ID NO:31～32；DYS576：SEQ ID NO:33～34；DYS570：SEQ ID NO:35～36；DYS481：SEQ ID NO:37～38；DYS627：SEQ ID NO:39～40；DYS460：SEQ ID NO:41～42；DYS449：SEQ ID NO:43～44；DYS533：SEQ ID NO:45～46；DYS518：SEQ ID NO:47～48；DYF387S1：SEQ ID NO:49～50。The kit of claim 1, wherein the primer pair sequences corresponding to the respective STR loci are as follows: DYS456: SEQ ID NO: 1 to 2; DYS458: SEQ ID NO: 3 to 4; DYS437: SEQ ID NO: 5 to 6 ; DYS439: SEQ ID NO: 7-8; DYS392: SEQ ID NO: 9-10; DYS385a/b: SEQ ID NO: 11-12; DYS393: SEQ ID NO: 13-14; DYS391: SEQ ID NO: 15. ～16; DYS390: SEQ ID NO: 17-18; DYS635: SEQ ID NO: 19-20; DYS438: SEQ ID NO: 21-22; DYS389I: SEQ ID NO: 23-24; DYS19: SEQ ID NO: 25. ～26; GATA-H4: SEQ ID NO: 27-28; DYS389II: SEQ ID NO: 29-30; DYS448: SEQ ID NO: 31-32; DYS576: SEQ ID NO: 33-34; DYS570: SEQ ID NO 35-36; DYS481: SEQ ID NO: 37-38; DYS627: SEQ ID NO: 39-40; DYS460: SEQ ID NO: 41-42; DYS449: SEQ ID NO: 43-44; DYS533: SEQ ID NO : 45 to 46; DYS518: SEQ ID NO: 47 to 48; DYF387S1: SEQ ID NO: 49 to 50.
- 权利要求1的试剂盒，其中每对引物中有一条引物的5’端用荧光染料标记。The kit of claim 1 wherein the 5' end of one of the primers in each pair of primers is labeled with a fluorescent dye.
- 权利要求3的试剂盒，其中使用所述荧光染料进行标记的方法为：DYS456、DYS576、DYS570、DYS481、DYF387S1、DYS627采用蓝色荧光染料FAM标记；DYS458、DYS460、DYS437、DYS439、DYS392、DYS385a/b采用绿色荧光染料HEX标记；DYS393、DYS391、DYS390、DYS635、DYS449、DYS533采用黄色荧光染料TAMRA标记；DYS438、DYS389I、DYS19、GATA-H4、DYS389II、DYS448和DYS518采用红色荧光染料ROX标记。The kit according to claim 3, wherein the method of labeling using the fluorescent dye is: DYS456, DYS576, DYS570, DYS481, DYF387S1, DYS627 are marked with a blue fluorescent dye FAM; DYS458, DYS460, DYS437, DYS439, DYS392, DYS385a/ b is labeled with green fluorescent dye HEX; DYS393, DYS391, DYS390, DYS635, DYS449, DYS533 are labeled with yellow fluorescent dye TAMRA; DYS438, DYS389I, DYS19, GATA-H4, DYS389II, DYS448 and DYS518 are marked with red fluorescent dye ROX.
- 权利要求1的试剂盒，其中所述试剂盒设有内标，所述内标选用橙色荧光染料Atto 633标记。The kit of claim 1 wherein said kit is provided with an internal standard, said internal standard being labeled with an orange fluorescent dye Atto 633.
- 权利要求1-5中任一项的试剂盒，其中所述试剂盒包括：无核酸酶水、PCR Master Mix、STRtyper-27Y Primer Mix、Allelic Ladder等位基因分型标准品、阳性对照品、Size-500橙色荧光分子量内标、光谱校正标准物。 The kit according to any one of claims 1 to 5, wherein the kit comprises: nuclease-free water, PCR Master Mix, STRtyper-27Y Primer Mix, Allelic Ladder allelic standard, positive control, Size -500 orange fluorescent molecular weight internal standard, spectral calibration standard.
- 权利要求6的试剂盒，其中所述PCR Master Mix包括DMSO 10mM、Tris-buffer 125mM、氯化钾125mM、硫酸铵65mM。The kit of claim 6 wherein said PCR Master Mix comprises DMSO 10 mM, Tris-buffer 125 mM, potassium chloride 125 mM, ammonium sulfate 65 mM.
- 权利要求6的试剂盒，其中所述STRtyper-27Y Primer Mix包括扩增所述STR基因座的所有引物、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。The kit of claim 6, wherein said STRtyper-27Y Primer Mix comprises all primers for amplifying said STR locus, deoxynucleotide triphosphate (dATP, dGTP, dTTP, dCTP) 7.5 mM, Taq enzyme 5 U/μl , magnesium chloride 7.5 mM, BSA 2.5 mg / ml.
- 权利要求8的试剂盒，其中所述引物的浓度如下：SEQ ID NO:1～2：0.84μM；SEQ ID NO:3～4：0.54μM；SEQ ID NO:5～6：0.3μM；SEQ ID NO:7～8：0.7μM；SEQ ID NO:9～10：1.36μM；SEQ ID NO:11～12：0.65μM；SEQ ID NO:13～14：0.5μM；SEQ ID NO:15～16：0.6μM；SEQ ID NO:17～18：0.65μM；SEQ ID NO:19～20：1.3μM；SEQ ID NO:21～22：1.5μM；SEQ ID NO:23～24：4.6μM；SEQ ID NO:25～26：1.9μM；SEQ ID NO:27～28：2.0μM；SEQ ID NO:29～30：2.7μM；SEQ ID NO:31～32：1.8μM；SEQ ID NO:33～34：0.5μM；SEQ ID NO:35～36：0.67μM；SEQ ID NO:37～38：0.66μM；SEQ ID NO:39～40：1.34μM；SEQ ID NO:41～42：1.26μM；SEQ ID NO:43～44：1.2μM；SEQ ID NO:45～46：1.3μM；SEQ ID NO:47～48：2.1μM；SEQ ID NO:49～50：0.67μM。 The kit of claim 8, wherein the concentration of said primer is as follows: SEQ ID NO: 1 to 2: 0.84 μM; SEQ ID NO: 3 to 4: 0.54 μM; SEQ ID NO: 5 to 6: 0.3 μM; NO: 7 to 8: 0.7 μM; SEQ ID NO: 9 to 10: 1.36 μM; SEQ ID NOs: 11 to 12: 0.65 μM; SEQ ID NOs: 13 to 14: 0.5 μM; SEQ ID NOs: 15 to 16: 0.6 μM; SEQ ID NOs: 17 to 18: 0.65 μM; SEQ ID NOs: 19 to 20: 1.3 μM; SEQ ID NOs: 21 to 22: 1.5 μM; SEQ ID NOs: 23 to 24: 4.6 μM; SEQ ID NO : 25 to 26: 1.9 μM; SEQ ID NOs: 27 to 28: 2.0 μM; SEQ ID NOs: 29 to 30: 2.7 μM; SEQ ID NOs: 31 to 32: 1.8 μM; SEQ ID NOs: 33 to 34: 0.5 μM; SEQ ID NO: 35-36: 0.67 μM; SEQ ID NO: 37-38: 0.66 μM; SEQ ID NO: 39-40: 1.34 μM; SEQ ID NO: 41-42: 1.26 μM; SEQ ID NO: 43-44: 1.2 μM; SEQ ID NOs: 45-46: 1.3 μM; SEQ ID NOs: 47-48: 2.1 μM; SEQ ID NOs: 49-50: 0.67 μM.
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| WO2020138995A1 (en) * | 2018-12-27 | 2020-07-02 | 주식회사 엔젠바이오 | Method for analyzing y str loci of human subject by using multiplex system, and analysis kit using same |
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| CN104694621A (en) * | 2014-10-29 | 2015-06-10 | 宁波海尔施基因科技有限公司 | Y-chromosome STR gene locus fluorescence labelled multiplex amplification kit with reinforcing resolution capability and application thereof |
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| CN110066877A (en) * | 2018-01-23 | 2019-07-30 | 深圳华大法医科技有限公司 | Y chromosome STR fluorescence labeling composite amplification system and kit |
| CN109750110B (en) * | 2019-03-07 | 2022-12-23 | 基点认知技术(北京)有限公司 | Composite amplification kit for 47 human autosomal and Y chromosome loci and application thereof |
| CN109880912B (en) * | 2019-03-07 | 2022-08-09 | 基点认知技术(北京)有限公司 | Composite amplification kit for 44 human Y chromosome loci and application thereof |
| CN112852971A (en) * | 2020-12-31 | 2021-05-28 | 百特元生物科技（北京）有限公司 | Primer group and kit for simultaneously amplifying 44 human Y-STR loci and application of primer group and kit |
| CN113584179A (en) * | 2021-04-14 | 2021-11-02 | 南方医科大学 | Six-color fluorescence labeling detection system for degrading 64 loci on human autosome and sex chromosome of test material typing |
| WO2023062409A1 (en) * | 2021-10-13 | 2023-04-20 | Dubai Police General Headquarters | Method and kit for multiplex analysis of rapidly mutating y-chromosome short tandem repeats |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102725422A (en) * | 2009-09-11 | 2012-10-10 | 生命科技公司 | Analysis of Y-chromosome STR markers |
| CN104017895A (en) * | 2014-06-24 | 2014-09-03 | 基点认知技术(北京)有限公司 | Composite amplification kit for 26 Y chromosome short tandem repeats |
| CN104694621A (en) * | 2014-10-29 | 2015-06-10 | 宁波海尔施基因科技有限公司 | Y-chromosome STR gene locus fluorescence labelled multiplex amplification kit with reinforcing resolution capability and application thereof |
-
2014
- 2014-10-29 CN CN201410591324.7A patent/CN104694621A/en active Pending
-
2015
- 2015-10-26 WO PCT/CN2015/092837 patent/WO2016066070A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102725422A (en) * | 2009-09-11 | 2012-10-10 | 生命科技公司 | Analysis of Y-chromosome STR markers |
| CN104017895A (en) * | 2014-06-24 | 2014-09-03 | 基点认知技术(北京)有限公司 | Composite amplification kit for 26 Y chromosome short tandem repeats |
| CN104694621A (en) * | 2014-10-29 | 2015-06-10 | 宁波海尔施基因科技有限公司 | Y-chromosome STR gene locus fluorescence labelled multiplex amplification kit with reinforcing resolution capability and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10745753B2 (en) | 2013-07-03 | 2020-08-18 | Government Of The United States Of America, As Represented By The Secretary Of Commerce | Mouse cell line authentication |
| WO2020138995A1 (en) * | 2018-12-27 | 2020-07-02 | 주식회사 엔젠바이오 | Method for analyzing y str loci of human subject by using multiplex system, and analysis kit using same |
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| CN104694621A (en) | 2015-06-10 |
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