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WO2016066070A1 - Trousse d'amplification multiplexe d'étiquetage fluorescent du locus str du chromosome y ayant une capacité d'identification améliorée et son utilisation - Google Patents

Trousse d'amplification multiplexe d'étiquetage fluorescent du locus str du chromosome y ayant une capacité d'identification améliorée et son utilisation Download PDF

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WO2016066070A1
WO2016066070A1 PCT/CN2015/092837 CN2015092837W WO2016066070A1 WO 2016066070 A1 WO2016066070 A1 WO 2016066070A1 CN 2015092837 W CN2015092837 W CN 2015092837W WO 2016066070 A1 WO2016066070 A1 WO 2016066070A1
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seq
kit
nos
loci
fluorescent dye
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Chinese (zh)
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李政
张兹钧
吴勇
金海英
宋丹璐
余丁
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NINGBO HEALTH GENE TECHNOLOGIES Co Ltd
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NINGBO HEALTH GENE TECHNOLOGIES Co Ltd
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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

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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

La présente invention concerne une trousse d'amplification multiplexe d'étiquetage fluorescent du locus STR du chromosome Y ayant une capacité d'identification améliorée, ainsi que son utilisation pour détecter les gènes d'ADN, ce qui permet d'amplifier et analyser les 27 loci STR suivants : 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 et DYS518.
PCT/CN2015/092837 2014-10-29 2015-10-26 Trousse d'amplification multiplexe d'étiquetage fluorescent du locus str du chromosome y ayant une capacité d'identification améliorée et son utilisation Ceased WO2016066070A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2020138995A1 (fr) * 2018-12-27 2020-07-02 주식회사 엔젠바이오 Procédé d'analyse de loci y str d'un sujet humain au moyen d'un système multiplex et kit d'analyse l'utilisant
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
EP4416309A4 (fr) * 2021-10-13 2025-10-15 Dubai Police General Headquarters Procédé et kit pour l'analyse multiplex des répétitions en tandem courtes du chromosome y à mutation rapide

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CN104694621A (zh) * 2014-10-29 2015-06-10 宁波海尔施基因科技有限公司 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及其应用
CN105177146B (zh) * 2015-09-21 2018-12-25 无锡中德美联生物技术有限公司 人类y染色体27个str基因座的荧光标记复合扩增试剂盒及其应用
CN106755414B (zh) * 2016-12-23 2020-09-01 宁波海尔施基因科技有限公司 一种检测dna遗传标记的方法
CN106834456B (zh) * 2017-01-16 2021-12-21 江苏苏博生物医学股份有限公司 一种采用荧光标记方法标记的y-str复合扩增检测试剂盒及其使用方法
CN107841566B (zh) * 2017-12-13 2021-11-05 苏州阅微基因技术有限公司 快速突变y染色体短串联重复序列的复合扩增体系、试剂盒及应用
CN110066877A (zh) * 2018-01-23 2019-07-30 深圳华大法医科技有限公司 Y染色体str荧光标记复合扩增系统及试剂盒
CN109880912B (zh) * 2019-03-07 2022-08-09 基点认知技术(北京)有限公司 44个人类y染色体基因座的复合扩增试剂盒及其应用
CN109750110B (zh) * 2019-03-07 2022-12-23 基点认知技术(北京)有限公司 47个人类常染色体与y染色体基因座的复合扩增试剂盒及其应用
CN112852971A (zh) * 2020-12-31 2021-05-28 百特元生物科技（北京）有限公司 一种同时扩增人44个y-str基因座的引物组、试剂盒及其应用
CN113584179A (zh) * 2021-04-14 2021-11-02 南方医科大学 一种用于降解检材分型的人类常染色体和性染色体上64个基因座的六色荧光标记检测体系

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CN104017895A (zh) * 2014-06-24 2014-09-03 基点认知技术(北京)有限公司 26个y染色体短串联重复序列的复合扩增试剂盒
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Cited By (3)

* Cited by examiner, † Cited by third party
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 (fr) * 2018-12-27 2020-07-02 주식회사 엔젠바이오 Procédé d'analyse de loci y str d'un sujet humain au moyen d'un système multiplex et kit d'analyse l'utilisant
EP4416309A4 (fr) * 2021-10-13 2025-10-15 Dubai Police General Headquarters Procédé et kit pour l'analyse multiplex des répétitions en tandem courtes du chromosome y à mutation rapide

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