CN106755448A - 29 fluorescence labeling composite amplification kits of str locus seat of human Y-chromosome - Google Patents
29 fluorescence labeling composite amplification kits of str locus seat of human Y-chromosome Download PDFInfo
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- CN106755448A CN106755448A CN201710004643.7A CN201710004643A CN106755448A CN 106755448 A CN106755448 A CN 106755448A CN 201710004643 A CN201710004643 A CN 201710004643A CN 106755448 A CN106755448 A CN 106755448A
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Abstract
The invention discloses a kind of 29 fluorescence labeling composite amplification kits of str locus seat of human Y-chromosome, its feature is：Described kit includes 29 pairs of specificity amplification primers of str locus seat, and wherein str locus seat is:DYS389Ⅰ/Ⅱ、DYS458、DYS19、DYS385a/b、DYS393、DYS391、DYS439、DYS635、DYS392、Y GATA H4、DYS437、DYS438、DYS448、DYS456、DYS390、DYS449、DYS576、DYS481、DYS549、DYS533、DYS570、DYS643、DYS460、DYS627、DYS387a/b、DYS518.The present invention has the advantages that the risk that cumulative individual discrimination is higher, effectively reduce allele crosses the border.
Description
Technical field
The present invention relates to molecular genetics field, a kind of specifically 29 fluorescence of str locus seat of human Y-chromosome
Mark composite amplification reagent kit.
Background technology
Tandem repeat loci (Short Tandem Repeat, STR) genetic marker also known as microsatellite DNA,
It is widely present in protokaryon and eukaryotic gene group, be a class by 2-6 nucleotides for repeat unit constitute tens to
The trinucleotide repeat sequence of more than 100, different number of core sequence is in middle repeated arrangement, and length is presented polymorphism, root
According to two ends conservative sequence, primer is designed, enter performing PCR, then by polyacrylamide, agarose gel electrophoresis or capillary
Electrophoresis etc. detect STR genetic markers polymorphism, STR mark compared with other genetic markers, str locus seat fragment it is small,
Easily amplification, is more suitable for micro and degraded sample, the amplification condition of each locus it is close and can composite amplification, thus have
Quickly, efficiently, it is accurate, sensitive, the advantages of contain much information, especially in terms of DNA databases are set up, with the technology such as conventional RFLP
Compare, STR composite amplification technologies have great superiority.
Y chromosome STR genetic markers refer to the STR for being present in human Y-chromosome non-recombinant region, mesh
Before, oneself has more than 220 to the Y-STR locus for confirming in a variety of manners and naming, the Y chromosome STR genetic markers that oneself is had found
Compared with euchromosome STR genetic marker, most of Y chromosome STR genetic markers have complicated repetitive structure, containing two kinds or
Two or more different recurring units, the core repeat sequence or number of repetition of individual in population is different, forms population genetic many
There is state property, Y chromosome STR genetic markers peculiar male, paternal inheritance and haplotype to make the three big features such as biography, and it is unique
It is combined with the superiority of STR bit point parting detection, medical jurisprudence individual identification, paternity test and DNA family trees structure etc. can be carried out.
The upper locus by mutation rate higher than 1 % of legal medical expert is referred to as rapid mutation locus (Rapidly Mutating Y-
STR), rapid mutation locus has related male's separating capacity higher, and typically has polymorphism higher, but base
The Y-STR familys of some situations can be disturbed Check is arranged because seat mutation rate is higher.
Carry out using Y-STR building storehouse on current legal medical expert, major function is family row Check, reduces suspect's scope, rather than recognize
Determine suspect, in China, particularly rural area, the mode that mainly family lives in concentrated communities, in the actual mechanical process that Y-STR builds storehouse,
Often only carrying out sampling to one or several members in an extended familys builds storehouse, just can profit so in the case of cost is less
Check scopes are detectd with Y-STR rapid drops, then by comparing euchromosome STR, suspect is assert, because Y-STR builds
One or several people of storehouse Bian samples are the Y-STR information for representing whole family, and this there is a kind of unfavorable Qing Condition：Suspect
Belong to this family a member, but there are 3 or more locus partings inconsistent with storehouse object Y-STR is built, now easily by suspect
Affiliated true family is excluded, if it is more to build rapid mutation locus in the kit of storehouse, then this possibility can become non-
Chang Gao, is not to assert suspect because Y-STR builds storehouse main purpose, it is not necessary to distinguish related male on the other hand, thus soon
Use is little herein for the advantage of fast mutator seat, so, it is not recommended that the gene of rapid mutation rate is used in this case
Seat, however, low mutation rate locus often polymorphism is relatively low, be unfavorable for the differentiation between family, accordingly, it would be desirable to locus
Quantity is more, while the polymorphism of locus is high, to ensure that Y-STR reagent gold utensils have sufficiently high overall polymorphism.
The commercial kit of main flow generally uses five fluorescent technique technology in the market, but with Y-STR
The expansion in Jian Ku markets, existing Y-STR kits cannot meet the demand of existing market, and the individuality of market in urgent need one is not
Rate is higher, detects the more Y-STR kits of locus number, is the Potential that necessarily becomes of STR fluorescence detection reagent kits.
On the market main flow kit have Yfiler, Yfiler Plus, PowerPlexY23, Goldeneye Y26,
The fluorescence detection reagent kits such as STRtyper27Y, AGCU Database Y24, locus quantity be respectively 17,27,23,
26,27,24, the locus negligible amounts of the conventional Yfiler of medical jurisprudence, cumulative individual discrimination is relatively low, Yfiler
Be identified in some specific crowds have low-down polymorphism, Yfiler P1us, STRtyper 27Y,
Powerplex Y23, Goldeneye Y26 contain 7,7,2 and 3 rapid mutation genes respectively.
The content of the invention
The invention aims to solving that cumulative individual discrimination of the prior art is relatively low, being crossed the border with allele
Risk defect, there is provided a kind of fluorescence labeling composite amplification kits of 29 str locus of human Y-chromosome seat are come on solving
State problem.
The invention discloses a kind of 29 fluorescence labeling composite amplification kits of str locus seat of human Y-chromosome, it is special
Put and be：Described kit includes 29 pairs of specificity amplification primers of str locus seat, and wherein str locus seat is: DYS389
Ⅰ/Ⅱ、DYS458、DYS19、DYS385a/b、DYS393、DYS391、DYS439、DYS635、DYS392、Y GATA H4、
DYS437、DYS438、DYS448、DYS456、DYS390、DYS449、DYS576、DYS481、DYS549、DYS533、DYS570、
DYS643、DYS460、DYS627、DYS387a/b、DYS518。
Preferably, in one embodiment of the invention, for above-mentioned 29 locus its repetitive sequence flank
Separately design specificity fluorescent labeled primer.
Preferably, the specificity amplification primer of described 26 pairs of str locus seat is divided into five groups:First group, DYS392,
DYS389I、DYS449、DYS389II、DYS438、DYS526、DYS570；Second group, DYS391, DYS456, DYS19,
DYS448、DYS385a、DYS385b；3rd group, DYS481, DYS437, DYS390, DYS460, DYS533, DYS458；4th
Group, DYS393, Y GATA H4, DYS439, DYS635, DYS570, DYS643；5th group, DYS549, DYS627, DYS387a,
DYS387b、DYS518；At least one using its 5' end of fluorochrome label in each pair primer.
Preferably, the fluorescence labeling composite amplification kit of described 29 str locus seats of human Y-chromosome is included instead
Mixed liquor, the specificity amplification primer of 26 pairs of str locus seat, the allele of 29 locus, hot start Taq polymerase, DNA is answered to mark
Quasi- product, sdH20 and fluorescence molecule amount internal standard, specific composition is：
| Reaction mixture | Containing Tris-HCl, MgCl2, KCl and dNTP Mix | 2ml |
| Database Y29 Primers | Containing 29 locus fluorescent primers | 1ml |
| Hot start Taq polymerase | Archaeal dna polymerase with heat endurance | 200ul |
| DNA standard items | Human genome DNA | 20ul |
| sdH 2O | Ultra-pure water | 1.85ml |
| Data base Y29 Allelic Ladder | Containing 29 allele of locus | 25ul |
| Marker SIZ-500 | Fluorescence molecule amount internal standard | 250ul |
Preferably, the composition of described reaction mixture is：MgCl27.5mM, Tris-HCl 125mM, KCl 125mM,
DNTPs7.5mM, BSA 2g/L.
Preferably, being combined the invention also discloses using the fluorescence labeling of 29 str locus seats of above-mentioned human Y-chromosome
Detection application of the amplification kit in forensic identification, paternity identification and DNA family trees build, specific detection method is：By reagent
Reagent loads PCR amplification pipes with sample DNA in box, is placed on thermal cycler, enters performing PCR amplification, then enters under conditions of 4 DEG C
Row insulation, obtains amplified production, amplified production is carried out into fluoroscopic examination on genetic analyzer, then use fragment analysis software
Detected on GeneMapper analysis gene sequencers, that is, obtained testing result.
Preferably, the amplification of Y chromosome str locus seat uses PCR, the detection of its amplified production is adopted
With Capillary Electrophoresis, polyacrylamide or agarose gel electrophoresis, the amplification program of PCR is：95 DEG C of 2min,
94 DEG C of 30S, 59 DEG C of 1min, 72 DEG C of 1min, totally 29 circulations；60℃ 30min.
Preferably, described sample DNA is using any one in Che1ex methods, magnetic bead extraction method or Organic extraction method
The human gene group DNA that the method for kind is extracted, or received using any one sanction body in hands-free take, filter paper, FTA cards, cotton swab, gauze
The human blood or Stomatocyte of collection.
Preferably, the source of described sample include the blood of people, blood stain, seminal fluid, saliva, body fluid, hair, muscle or
Histoorgan.
The present invention has advantages below compared to existing technology：
（1）ANLONG Y29 possess 29 low mutation rate locus, be on the market the most Y-STR kits of locus quantity it
One；
（2）By five fluorescent technique system detectio obtain it is a kind of there is high-resolution, low mutation rate and disclosure satisfy that Y-STR builds
The demand that storehouse operation family is quickly investigated；
（3）Not only locus is more for ANLONG Y29, and the allele scope more great , Minus of locus Marker coverings are few
The risk that allele crosses the border, and otherwise performance also has larger improvement.
Specific embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed implementation method and specific operating process, but protection scope of the present invention is not limited to following implementations
Example.
The invention discloses a kind of 29 fluorescence labeling composite amplification kits of str locus seat of human Y-chromosome, it is special
Put and be：Described kit includes 29 pairs of specificity amplification primers of str locus seat, and wherein str locus seat is: DYS389
Ⅰ/Ⅱ、DYS458、DYS19、DYS385a/b、DYS393、DYS391、DYS439、DYS635、DYS392、Y GATA H4、
DYS437、DYS438、DYS448、DYS456、DYS390、DYS449、DYS576、DYS481、DYS549、DYS533、DYS570、
DYS643、DYS460、DYS627、DYS387a/b、DYS518。
Preferably, in one embodiment of the invention, for above-mentioned 29 locus its repetitive sequence flank
Separately design specificity fluorescent labeled primer.
Preferably, the specificity amplification primer of described 26 pairs of str locus seat is divided into five groups:First group, DYS392,
DYS389I、DYS449、DYS389II、DYS438、DYS526、DYS570；Second group, DYS391, DYS456, DYS19,
DYS448、DYS385a、DYS385b；3rd group, DYS481, DYS437, DYS390, DYS460, DYS533, DYS458；4th
Group, DYS393, Y GATA H4, DYS439, DYS635, DYS570, DYS643；5th group, DYS549, DYS627, DYS387a,
DYS387b、DYS518；At least one using its 5' end of fluorochrome label in each pair primer.
Preferably, the fluorescence labeling composite amplification kit of described 29 str locus seats of human Y-chromosome is included instead
Mixed liquor, the specificity amplification primer of 26 pairs of str locus seat, the allele of 29 locus, hot start Taq polymerase, DNA is answered to mark
Quasi- product, sdH20 and fluorescence molecule amount internal standard, specific composition is：
| Reaction mixture | Containing Tris-HCl, MgCl2, KCl and dNTP Mix | 2ml |
| Database Y29 Primers | Containing 29 locus fluorescent primers | 1ml |
| Hot start Taq polymerase | Archaeal dna polymerase with heat endurance | 200ul |
| DNA standard items | Human genome DNA | 20ul |
| sdH 2O | Ultra-pure water | 1.85ml |
| Data base Y29 Allelic Ladder | Containing 29 allele of locus | 25ul |
| Marker SIZ-500 | Fluorescence molecule amount internal standard | 250ul |
Preferably, the composition of described reaction mixture is：MgCl27.5mM, Tris-HCl 125mM, KCl 125mM,
DNTPs7.5mM, BSA 2g/L.
Preferably, being combined the invention also discloses using the fluorescence labeling of 29 str locus seats of above-mentioned human Y-chromosome
Detection application of the amplification kit in forensic identification, paternity identification and DNA family trees build, specific detection method is：By reagent
Reagent loads PCR amplification pipes with sample DNA in box, is placed on thermal cycler, enters performing PCR amplification, then enters under conditions of 4 DEG C
Row insulation, obtains amplified production, amplified production is carried out into fluoroscopic examination on genetic analyzer, then use fragment analysis software
Detected on GeneMapper analysis gene sequencers, that is, obtained testing result.
Preferably, the amplification of Y chromosome str locus seat uses PCR, the detection of its amplified production is adopted
With Capillary Electrophoresis, polyacrylamide or agarose gel electrophoresis, the amplification program of PCR is：95 DEG C of 2min,
94 DEG C of 30S, 59 DEG C of 1min, 72 DEG C of 1min, totally 29 circulations；60℃ 30min.
Preferably, described sample DNA is using any one in Che1ex methods, magnetic bead extraction method or Organic extraction method
The human gene group DNA that the method for kind is extracted, or received using any one sanction body in hands-free take, filter paper, FTA cards, cotton swab, gauze
The human blood or Stomatocyte of collection.
Preferably, the source of described sample include the blood of people, blood stain, seminal fluid, saliva, body fluid, hair, muscle or
Histoorgan.
Embodiment 1
The screening of Y chromosome str locus seat
By to DYS392, DYS389I, DYS389II, DYS531, DYS630, DYS447, DYS438, DYS527a,
DYS527b、DYS522、DYS391、DYS456、DYS19、DYS622、DYS388、DYS448、DYS385a、DYS385b、
DYS443、DYS446、DYS 552、DYS 510、DYS 630、DYS587、DYS481、DYS437、DYS390、DYS460、
DYS533、DYS458、DYS393、Y GATA H4、DYS439、DYS635、DYS444、DYS643、DYS549、DYS557、
DYS643, DYS607, DYS 525, DYS576, DYS520 DYS557, YGATA-A10 totally 45 Y chromosome Short tandem repeatSTRs
Sequence gene locus and its allele hereditary feature have made intensive studies, and filter out 29 Y chromosome STRs
Locus：DYS389Ⅰ/Ⅱ、DYS458、DYS19、DYS385a/b、DYS393、DYS391、DYS439、DYS635、DYS392、Y
GATA H4、DYS437、DYS438、DYS448、DYS456、DYS390、DYS449、DYS576、DYS481、DYS549、
DYS533、DYS570、DYS643、DYS460、DYS627、DYS387a/b、DYS518.This 29 Y chromosome Short tandem repeatSTR sequences
Row locus has the good gene frequency distribution of relatively low genetic polymorphism box in crowd, meet the present invention for
The requirement in site.
Embodiment 2
This kit is since it is desired that meet the adaptability of different samples and the tolerance of different annealing temperature, so needing test not
Same amplification program, ensures that different samples can obtain correct DNA typing within the scope of temperature wider.
First, period test：28 circulations are tested respectively, and 29 circulations, 30 circulations, 31 circulations are most preferably followed
Ring is 29；Secondly, annealing temperature test：56 DEG C are tested respectively, and 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 62 DEG C, optimum temperature is 59
Degree.
Finally amplification program is：
95 DEG C of 2min, 94 DEG C of 30S, 59 DEG C of 1min, 72 DEG C of 1min 29 are circulated；60 DEG C of 30min, 4 DEG C of extensions.
The method of this kit detection amplified production is to be measured using capillary genetic analyzer or gel electrophoresis.
General principle of the invention, principal character and advantages of the present invention has been shown and described above.The technology of the industry
Personnel it should be appreciated that the present invention is not limited to the above embodiments, the simply present invention described in above-described embodiment and specification
Principle, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these change and
Improvement is both fallen within the range of claimed invention.The protection domain of application claims by appending claims and its
Equivalent is defined.
Claims (9)
1. fluorescence labeling composite amplification kits of 29 str locus of a kind of human Y-chromosome seat, it is characterised in that：Described
Kit includes 29 pairs of specificity amplification primers of str locus seat, and wherein str locus seat is:DYS389Ⅰ/Ⅱ、DYS458、
DYS19、DYS385a/b、DYS393、DYS391、DYS439、DYS635、DYS392、Y GATA H4、DYS437、DYS438、
DYS448、DYS456、DYS390、DYS449、DYS576、DYS481、DYS549、DYS533、DYS570、DYS643、DYS460、
DYS627、DYS387a/b、DYS518。
2. fluorescence labeling composite amplification reagents of 29 str locus of a kind of human Y-chromosome according to claim 1 seat
Box, it is characterised in that：For above-mentioned 29 locus specificity fluorescent labeled primer is separately designed in the flank of its repetitive sequence.
3. fluorescence labeling composite amplification reagents of 29 str locus of a kind of human Y-chromosome according to claim 1 seat
Box, it is characterised in that：The specificity amplification primer of described 26 pairs of str locus seat is divided into five groups:First group, DYS392,
DYS389I、DYS449、DYS389II、DYS438、DYS526、DYS570；Second group, DYS391, DYS456, DYS19,
DYS448、DYS385a、DYS385b；3rd group, DYS481, DYS437, DYS390, DYS460, DYS533, DYS458；4th
Group, DYS393, Y GATA H4, DYS439, DYS635, DYS570, DYS643；5th group, DYS549, DYS627, DYS387a,
DYS387b、DYS518；At least one using its 5' end of fluorochrome label in each pair primer.
4. fluorescence labeling composite amplification reagents of 29 str locus of a kind of human Y-chromosome according to claim 1 seat
Box, it is characterised in that：The fluorescence labeling composite amplification kit of described 29 str locus seats of human Y-chromosome includes reaction
Mixed liquor, the specificity amplification primer of 26 pairs of str locus seat, allele, hot start Taq polymerase, the DNA standards of 29 locus
Product, sdH20 and fluorescence molecule amount internal standard, specific composition is：
5. fluorescence labeling composite amplification reagents of 29 str locus of a kind of human Y-chromosome according to claim 1 seat
Box, it is characterised in that：The composition of described reaction mixture is：MgCl27.5mM, Tris-HCl 125mM, KCl 125mM,
DNTPs7.5mM, BSA 2g/L.
6. any described 29 fluorescence labeling composite amplifications of str locus seat of human Y-chromosome of a kind of use claim 1-5
Detection method of the kit in forensic identification, paternity identification and DNA family trees build, it is characterised in that：Specifically detection method is：
Reagent in kit and sample DNA are loaded into PCR amplification pipes, is placed on thermal cycler, enter performing PCR amplification, then in 4 DEG C of bar
It is incubated under part, is obtained amplified production, amplified production is carried out into fluoroscopic examination on genetic analyzer, then it is soft with fragment analysis
Detected on part GeneMapper analysis gene sequencers, that is, obtained testing result.
7. a kind of detection method according to claim 6, it is characterised in that：The amplification of Y chromosome str locus seat is using poly-
Polymerase chain reacts, and the detection of its amplified production uses Capillary Electrophoresis, polyacrylamide or agarose gel electrophoresis, polymerase
The amplification program of chain reaction is：95 DEG C of 2min, 94 DEG C of 30S, 59 DEG C of 1min, 72 DEG C of 1min, totally 29 circulations；60℃30min.
8. a kind of detection method according to claim 6, it is characterised in that：Described sample DNA is using Che1ex
Method, magnetic bead extraction method or in Organic extraction method any one method extract human gene group DNA, or using hands-free take, filter paper,
Any one cuts out the human blood or Stomatocyte that body is collected in FTA cards, cotton swab, gauze.
9. a kind of detection method according to claim 6, it is characterised in that：The source of described sample includes the blood of people
Liquid, blood stain, seminal fluid, saliva, body fluid, hair, muscle or histoorgan.
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| CN108060233A (en) * | 2017-12-13 | 2018-05-22 | 苏州阅微基因技术有限公司 | Fluorescent composite amplification system, kit and the application of 30 Y chromosome str locus seats |
| CN108330177A (en) * | 2017-10-26 | 2018-07-27 | 杭州祥音生物医药科技有限公司 | Identify human Y-chromosome parting primer sets and its application and apply its product |
| CN109880912A (en) * | 2019-03-07 | 2019-06-14 | 基点认知技术(北京)有限公司 | The composite amplification reagent kit of 44 human Y-chromosome locus and its application |
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| CN112143816A (en) * | 2019-06-26 | 2020-12-29 | 司法鉴定科学研究院 | 29-plex Y-STR typing system for family search and paternal biological geographic ancestry inference |
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2017
- 2017-01-04 CN CN201710004643.7A patent/CN106755448A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108330177A (en) * | 2017-10-26 | 2018-07-27 | 杭州祥音生物医药科技有限公司 | Identify human Y-chromosome parting primer sets and its application and apply its product |
| CN108060233A (en) * | 2017-12-13 | 2018-05-22 | 苏州阅微基因技术有限公司 | Fluorescent composite amplification system, kit and the application of 30 Y chromosome str locus seats |
| CN108060233B (en) * | 2017-12-13 | 2021-04-30 | 苏州阅微基因技术有限公司 | Fluorescence multiplex amplification system and kit for 30Y chromosome STR loci and application |
| CN109929936A (en) * | 2019-03-06 | 2019-06-25 | 广东华美众源生物科技有限公司 | It is a kind of detect human Y-chromosome rapid mutation str locus seat fluorescence labeling composite amplification kit and application |
| CN109929936B (en) * | 2019-03-06 | 2023-03-10 | 广东华美众源生物科技有限公司 | Fluorescence labeling multiplex amplification kit for detecting human Y chromosome rapid mutation STR locus and application |
| CN109880912A (en) * | 2019-03-07 | 2019-06-14 | 基点认知技术(北京)有限公司 | The composite amplification reagent kit of 44 human Y-chromosome locus and its application |
| 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 |
| CN112143816A (en) * | 2019-06-26 | 2020-12-29 | 司法鉴定科学研究院 | 29-plex Y-STR typing system for family search and paternal biological geographic ancestry inference |
| CN112143816B (en) * | 2019-06-26 | 2023-12-01 | 司法鉴定科学研究院 | 29-plex Y-STR typing system for family search and paternal biological geographic ancestor inference |
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Application publication date: 20170531 |