CN110423802A - Composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat - Google Patents
Composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat Download PDFInfo
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- CN110423802A CN110423802A CN201910784935.6A CN201910784935A CN110423802A CN 110423802 A CN110423802 A CN 110423802A CN 201910784935 A CN201910784935 A CN 201910784935A CN 110423802 A CN110423802 A CN 110423802A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present invention discloses composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat, and including at least the specificity amplification primer pair of 40 locus, sequence includes NO.1~78 SEQ ID.The application can detect 20 Y chromosome str locus seats, 19 euchromosome STR locus and Amelogenin locus simultaneously, and not only parting is accurate, but also shorten detection time, improve exclusion and determine suspicion of crime human efficiency.
Description
Technical field
The invention belongs to analyze genetic arts, it is related to the composite amplification checking system of human chromosomal str locus seat, especially
It is related to composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat.
Background technique
For DNA-STR typing method due to having specific good, high sensitivity, parting is easy quickly, and it is excellent to be easy to standardization etc.
Point has become one of the main means of legal medical material evidence examination since the 1990s.Currently used STR detection kit
Including euchromosome STR detection kit and Y chromosome STR detection kit, individual identification detection and father can be carried out respectively
System's detection.When a major criminal cases occur, the material evidence often extracted to scene first carries out euchromosome STR detection,
When being confirmed as material evidence that male offense suspect leaves, then plus do Y chromosome STR detection, thus increase investigation clue.
The Y chromosome STR kit (Yfiler Plus) of most commonly used ABI company production domestic at present and often dyeing
Body STR kit (Identifiler) because being influenced by sample state, amplification site number, the factors such as amplification system, only into
Then time-consuming 5-6 hour, check problem so twice often prolong because detection time is increased row twice PCR amplification procedure
The accidentally killer opportunity of crime suspect, while as suspect leaves material evidence amount at the scene causes to detect less
Failure, finally loses investigation clue.
To sum up, increasing with criminal case, it is right under battle conditions that existing detection kit can not meet public security well
In the quick requirement examined with field test ability of forensic dna, euchromosome STR locus and Y chromosome STR how are realized
Locus detects together, reaches and shortens detection time while improving exclusion and determining that suspicion of crime human efficiency is to be solved as having
The problem of.
Summary of the invention
In view of the problems of the above-mentioned prior art, autosome and Y are detected simultaneously the purpose of the present invention is to propose to a kind of
The composite amplification reagent kit of chromosome str locus seat.
The purpose of the invention will be achieved through the following technical solutions:
Composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat can include at least 40
The specificity amplification primer pair of a locus, sequence can respectively successively are as follows: and NO:1~2 SEQ ID, SEQ ID NO:3~
4, NO:5~6 SEQ ID, NO:7~8 SEQ ID, NO:9~10 SEQ ID, NO:11~12 SEQ ID, SEQ ID NO:13
~14, NO:15~16 SEQ ID, NO:17~18 SEQ ID, NO:19~20 SEQ ID, NO:21~22 SEQ ID, SEQ
NO:23~24 ID, NO:25~26 SEQ ID, NO:27~28 SEQ ID, NO:29~30 SEQ ID, SEQ ID NO:31
~32, NO:33~34 SEQ ID, NO:35~36 SEQ ID, NO:37~38 SEQ ID, NO:39~40 SEQ ID, SEQ
NO:41~42 ID, NO:43~44 SEQ ID, NO:45~46 SEQ ID, NO:47~49 SEQ ID, SEQ ID NO:50
~51, NO:52~53 SEQ ID, NO:54~55 SEQ ID, NO:56~57 SEQ ID, NO:58~59 SEQ ID, SEQ
NO:60~61 ID, NO:62~63 SEQ ID, NO:64~65 SEQ ID, NO:66~67 SEQ ID, SEQ ID NO:66
~68, NO:69~70 SEQ ID, NO:71~72 SEQ ID, NO:73~74 SEQ ID, NO:75~76 SEQ ID, SEQ
NO:77~78 ID.
In some embodiments, the specificity amplification primer can correspond to 40 str locus seats, 40 STR bases
Because seat can be located at least one autosome and at least one Y chromosome, the locus can be successively are as follows: DYS391,
D3S1358、D13S317、D7S820、D16S539、Penta E、DYS635、DYS576、DYS456、TPOX、TH01、
D2S1338、D6S1043、DYS385a/b、DYS438、DYS448、DYS437、D19S433、vWA、DYS19、CSF1PO、
D18S51、FGA、Amelogenin、D8S1179、D5S818、D21S11、D12S391、Penta D、DYS458、DYS533、
DYS393, DYS389I/II, DYS439, DYS392, Y_GATA_H4, DYS390 and DYS481.
In some embodiments, the final concentration of the specificity amplification primer can be 0.15 μM~0.3 μM.
In some embodiments, 40 locus can be grouped.
In some embodiments, 40 locus can at least be divided into 5 groups: DYS391, D3S1358,
D13S317, D7S820, D16S539, Penta E, DYS635, DYS576 are first group；DYS456,TPOX,TH01,
D2S1338, D6S1043, DYS385a/b, DYS438, DYS448 are second group；DYS437,D19S433,vWA,DYS19,
CSF1PO, D18S51, FGA are third group；Amelogenin,D8S1179,D5S818,D21S11,D12S391,Penta D,
DYS458, DYS533 are the 4th group；DYS393,DYS389I/II,DYS439,DYS392,Y_GATA_H4,DYS390,DYS481
It is the 5th group.
In some embodiments, 5 ' ends of at least one primer in the specificity amplification primer can be marked
Note.
In some embodiments, 5 ' ends of the corresponding specificity amplification primer of each group locus can be contaminated using fluorescence
Material label.
In some embodiments, the fluorescent dye may include 6-FAM, HEX, SUM, LYN, and/or PUR.
In some embodiments, the kit includes reaction mixture, specific composite amplification primer, thermal starting Taq
Enzyme, DNA standard items, sdH2O, Allelic Ladder, fluorescent molecule amount internal standard, the wherein composition of reaction mixture are as follows: Tris-
HCl 50mM、MgCl2 2.0mM、KCl 50mM、dNTPs 0.2mM、BSA 0.8mg/ml。
In some embodiments, the amplification system of the kit is reaction mixture: 10.0 μ L；Hot start Taq polymerase:
1.0μL；Primer mixture: 5.0 μ L；Genomic DNA: 0.125ng~5.0ng；sdH2O complements to 25.0 μ L.
A kind of application method according to above-mentioned composite amplification reagent kit, when the specificity amplification primer is for expanding
When, amplification program can be with are as follows: and 95 DEG C of 2min of denaturation recycle 94 DEG C of 30s, 60 DEG C of 40s, 72 DEG C of 1min 15 circulations, 90 DEG C of 30s,
58 DEG C of 1min, 65 DEG C of 1min20s 15 circulations, terminate and extend 60 DEG C of 20min, and 4 DEG C of heat preservations maintain.
It is a kind of to be reflected according to above-mentioned composite amplification reagent kit in legal medical expert's individual identification, suspect's family investigation, and/or parental right
Application in fixed.
In some embodiments, the composite amplification reagent kit can be adapted for a plurality of types of samples, the sample packet
Include using the human gene group DNA that any one method is extracted in Chelex method, magnetic bead extraction method or organic method extraction process, and/or
The human blood or Stomatocyte collected by any one carrier in filter paper, FTA card, cotton swab, gauze.
Compared with prior art, provided by the invention a kind of to detect answering for autosome and Y chromosome str locus seat simultaneously
Close amplification kit, reach have the technical effect that 1) the application can detect simultaneously 20 Y chromosome str locus seats, 19 often
Chromosome str locus seat and Amelogenin locus realize while detecting euchromosome STR locus and Y chromosome STR
The purpose of locus, not only parting is accurate, but also shortens detection time, improves exclusion and determine suspicion of crime human efficiency；2)
Autosome and Y chromosome str locus seat can be detected simultaneously based on the application, need separate detection autosome with existing
It is compared with the kit of Y chromosome str locus seat, can not only save amplification template consumption, but also can be to a certain degree
It is upper to solve the problems, such as micro sample in case；3) the application has very strong sample adaptability, i.e., a kind of kit can be used for expanding
Increase the sample of kind of sample；4) the application can satisfy public security under battle conditions for the quick inspection of forensic dna and field test ability
Requirement effective technical guarantee is provided.
Detailed description of the invention
Fig. 1 is the arrangement schematic diagram of the locus according to shown in the application some embodiments；
Fig. 2 is the allele Allelic Ladder parting figure according to shown in the application some embodiments；
Fig. 3 is the parting figure that the kit according to shown in the application some embodiments extracts sample；
Fig. 4 is the parting figure of the kit standard DNA 9948 according to shown in the application some embodiments.
Below just in conjunction with the embodiments, the embodiment of the present invention is described in further detail, so that technical solution is more
It should be readily appreciated that, grasp.
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.In following embodiments
The experimental method is unless otherwise specified conventional method；The reagent and material unless otherwise specified can be from business
Approach obtains, the scope of the patents that following example is not intended to limit the invention, all equivalence enforcements without departing from carried out by the present invention
Or change, it is intended to be limited solely by the scope of this patent.
The present invention provides composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat.Short string
Connection repetitive sequence (Short Tandem Repeat, STR) is that the one kind being present in human genome has length polymorphism
DNA sequence dna, core sequence are generally made of 2~6 bases, because the core sequence is in different number of string between Different Individual
Connection repeats and shows length polymorphism.Average every 6-10kb just has a str locus seat in human gene group DNA, these are extensively
Being distributed in the polymorphic repetitive sequence of height in human genome becomes effective genetic marker of individual identification and paternity identification, can use polymerization
The method of enzyme chain reaction (Polymerase Chain Reaction, PCR) and electrophoretic separation is detected.
Euchromosome STR locus and Y chromosome str locus seat in the present invention are 40 genes obtained by sieve series
Seat.It can be with described in reference implementation example 1, details are not described herein about screening step.In some embodiments, 40 locus
May include DYS391, D3S1358, D13S317, D7S820, D16S539, Penta E, DYS635, DYS576, DYS456,
TPOX、TH01、D2S1338、D6S1043、DYS385a/b、DYS438、DYS448、DYS437、D19S433、vWA、DYS19、
CSF1PO、D18S51、FGA、Amelogenin、D8S1179、D5S818、D21S11、D12S391、Penta D、DYS458、
DYS533, DYS393, DYS389I/II, DYS439, DYS392, Y_GATA_H4, DYS390 and DYS481.In some embodiments
In, 40 locus can be divided into two groups or more.Preferably, 40 locus can be divided into 5 groups.More preferably,
40 locus can be divided into following 5 groups: DYS391, D3S1358, D13S317, D7S820, D16S539, PentaE,
DYS635, DYS576 are first group；DYS456,TPOX,TH01,D2S1338,D6S1043,DYS385a/b,DYS438,
DYS448 is second group；DYS437, D19S433, vWA, DYS19, CSF1PO, D18S51, FGA are third group；Amelogenin,
D8S1179, D5S818, D21S11, D12S391, Penta D, DYS458, DYS533 are the 4th group；DYS393,DYS389I/
II, DYS439, DYS392, Y_GATA_H4, DYS390, DYS481 are the 5th group.
In some embodiments, the composite amplification reagent kit is to be used for while expanding euchromosome STR locus and Y dye
The corresponding specific primer of colour solid locus.The kit includes reaction mixture, specific composite amplification primer, thermal starting
Taq enzyme, DNA standard items, sdH2O, Allelic Ladder, fluorescent molecule amount internal standard, amplification system is reaction mixture: 10.0
μL；Hot start Taq polymerase: 1.0 μ L；Primer mixture: 5.0 μ L；Genomic DNA: 0.125ng~5.0ng；sdH2O is complemented to
25.0μL。
The specificity amplification primer can refer to the primer containing NO:1~78 SEQ ID (as shown in table 1), the SEQ
The concentration of the primer of NO:1~78 ID can be with 0.15 μM~0.3 μM.In some embodiments, SEQ ID NO:1~78
The concentration of primer can be concentration as shown in Table 1.In some embodiments, the specificity amplification primer corresponds to the present invention
40 locus, for example, its corresponding relationship can be shown in reference table 1.In some embodiments, specific amplification can be drawn
5 ' ends of at least one primer in object are marked.For example, can be at least one in specificity amplification primer shown in table 1
5 ' ends of primer are marked.Including but not limited to fluorescence can be selected when the specificity amplification primer is marked
Dye marker method etc..Wherein, fluorescent dye can include but is not limited to 6-FAM, HEX, SUM, LYN, PUR etc..In some implementations
In example, when 5 ' ends of at least two or more primers in specificity amplification primer are marked, the fluorescent dye mark of selection
Note object can be that identical, part is same or different.It preferably, can be by the corresponding locus point of specificity amplification primer
Several groups (for example, 2 groups, 3 groups, 4 groups, 5 groups), are then marked, wherein to each group gene coordinate according to each group locus
The fluorescent dye that note is selected can be that identical, part is identical or entirely different.It is highly preferred that can be by specific amplification
Corresponding 5 groups of the locus point of primer, the fluorochrome label object that each group locus label is selected are also entirely different.Particularly preferably
The corresponding locus of specificity amplification primer can be divided into 5 groups of locus as described in Example 2 by ground, and be selected strictly according to the facts
Apply the corresponding marker of each group locus shown in example 2.It can be with reference implementation example 2 and Fig. 1 institute about the arrangement of more locus
Show.
Reaction mixture can include but is not limited to Tris-HCl, MgCl2, KCl, dNTPs etc..Preferably, reaction mixing
Liquid may include Tris-HCl 50mM, MgCl2 2.0mM、KCl 50mM、dNTPs 0.2mM、BSA 0.8mg/ml。
Hot start Taq polymerase is a kind of archaeal dna polymerase with thermal stability, and the content of the archaeal dna polymerase is 5U/ μ L.
sdH2O is ultrapure water.
Allelic Ladder also known as allelic ladder, effect are to correct PCR product in electrophoresis process
The mobility deviation of generation, to guarantee to obtain correct measuring samples genotype.
Fluorescent molecule amount internal standard, i.e. AGCU Marker SIZ-600, are the mixtures comprising 34 DNA fragmentations, act on
In mark PCR product size.
In some embodiments, the kit can be applied to legal medical expert's individual identification, suspect's family investigation, parental right mirror
It is one of fixed or any combination thereof.When the kit is applied to legal medical expert's individual identification, suspect's family investigation, paternity identification
One of (for example, victim's remains identification in abducted children, disaster) or any combination thereof, genotyping result is correct.Tool
Body, it can be further illustrated by taking embodiment 6 as an example.Wherein, the merely illustrative example of purpose of embodiment 6 illustrates, is not intended to limit
The protection scope of the application.In some embodiments, the composite amplification reagent kit can be adapted for a plurality of types of samples, institute
Stating sample includes but is not limited to utilize Chelex method, the people that any one method is extracted in magnetic bead extraction method or organic method extraction process
Genomic DNA, and/or by filter paper, FTA card, cotton swab, gauze any one carrier collect human blood or oral cavity it is thin
Born of the same parents.
Further below to the detailed description of the invention in a manner of specific embodiment, following the description is not to the model of the application
It encloses and is construed as limiting.
The screening of 1 autosome of embodiment and Y chromosome str locus seat
By to DYS549, DYS522, DYS389I, DYS439, DYS447, DYS389II, DYS392, DYS576,
DYS19、DYS388、DYS391、DYS635、DYS460、DYS456、DYS533、DYS520、DYS438、DYS527a/b、
DYS449、DYS709、DYS622、DYS481、DYS437、DYS390、DYS630、DYS448、Y_GATA_H4、DYS458、
DYS607、DYS393、DYS552、DYS570、DYS385a/b、DYS593、DYS444、DYS643、Amelogenin、vWA、
D10S1248、D2S441、D21S11、D18S51、D5S818、D7S820、D13S317、D16S539、FGA、D2S1338、
D22S1045、D1S1656、D19S433、D6S1043、D12S391、D8S1179、D3S1358、CSF1PO、Penta D、
The research of PentaE, TH01 and TPOX totally 62 locus, it is ensured that selected locus and common kit are (for example, ABI company
Identifiler and Yfiler Plus) locus there is certain compatibility, finally filter out 20 Y chromosome STR bases
Because seat (for example, with " DYS " start locus title, including DYS391, DYS635, DYS576, DYS456, DYS385a/b,
DYS438、DYS448、DYS437、DYS19、DYS458、DYS533、DYS393、DYS389I/II、DYS439、DYS392、Y_
GATA_H4, DYS390 and DYS481), 19 euchromosome STR locus and Amelogenin locus.The compatibility can
To refer to respectively using the amplification system of the application (for example, the primer of the application, primer concentration, reaction mixture, reaction mixture
Concentration etc.) and amplification program, common agents box amplification system (for example, the primer of common agents box, primer concentration, reaction are mixed
Close liquid, reaction mixture concentration etc.) and amplification program parting is carried out to identical locus, then use the application and common agents
Its genotyping result of box is consistent.The purpose of the compatibility is can be shared and be exchanged with existing DNA data.More than being based on
Described, the locus filtered out is as follows: DYS391, D3S1358, D13S317, D7S820, D16S539, Penta E, DYS635,
DYS576、DYS456、TPOX、TH01、D2S1338、D6S1043、DYS385a/b、DYS438、DYS448、DYS437、
D19S433、vWA、DYS19、CSF1PO、D18S51、FGA、Amelogenin、D8S1179、D5S818、D21S11、D12S391、
Penta D, DYS458, DYS533, DYS393, DYS389I/II, DYS439, DYS392, Y_GATA_H4, DYS390 and
DYS481.Further, the details about each locus filtered out are as shown in table 1.
The relevant information of 1 40 locus of table
2 40 locus grouping arrangement situations of embodiment
DYS391, D3S1358, D13S317, D7S820, D16S539, Penta E, DYS635, DYS576 are first group,
Fluorochrome label object is 6-FAM；DYS456,TPOX,TH01,D2S1338,D6S1043,DYS385a/b,DYS438,
DYS448 is second group, and fluorochrome label object is HEX；DYS437,D19S433,vWA,DYS19,CSF1PO,D18S51,FGA
For third group, fluorochrome label object is SUM；Amelogenin,D8S1179,D5S818,D21S11,D12S391,Penta
D, DYS458, DYS533 are the 4th group, and fluorochrome label object is LYN；DYS393,DYS389I/II,DYS439,DYS392,
Y_GATA_H4, DYS390, DYS481 are the 5th group, and fluorochrome label object is PUR, and packet diagram is as shown in Figure 1.
The selection of 3 specific primer design of embodiment and primer concentration
With the increase of primer quantity in composite amplification system, interfering with each other also increasingly between different locus primer
Seriously, the dynamics of reaction system becomes to become increasingly complex, it is therefore desirable to design a large amount of primer sequence and carry out complicated test
And grope concentration ratio special between primer in composite amplification system, finally ensure that do not reduce kit specificity and it is sensitive
Compound more huamn autosomals and Y chromosome str locus seat in the case where degree.Specifically, including the following steps:
(1) specificity amplification primer designs
Before design primer, it is necessary to analyze the property of target sequence to be measured, select highly conserved, the uniform region of base distribution
Carry out design of primers.
A. primer Tm: since algorithm is different, the primer Tm of different software design is not quite similar, but needs as far as possible
Guarantee that the Tm value of upstream and downstream primer is consistent, is usually no more than 2 DEG C.
B. it dimer between primer: avoids continuing to exceed 3 base pair complementarities between two 3 ' ends of primer as far as possible.
C. primer and template sequence non-specific binding: avoid primer sequence non-specific with template sequence as far as possible in design process
Region combines.
D. primer compares: primer sequence needs to carry out blast comparison using NCBI, other than specific primer combined area,
The fewer matching area the better.
(2) primer is verified
Firstly, carrying out the single of primer expands experiment, exclusion has primer non-specific, that amplification efficiency is low；Secondly, carrying out monochromatic small
Multiple to expand experiment, i.e., by each group of primer, individually hybrid test needs to exclude to generate non-specific and amplification effect as single expand
The low primer of rate；It is tested finally, carrying out big multiple expand, i.e. four groups of primer whole hybrid tests, is primarily determining that each locus is multiple
After closing amplimer, tentatively assembling for primer is carried out, is template using standard DNA and other practical DNA samples, is investigated each
The concentration and detection efficiency of locus primer investigate non-specific amplification product, and if it exists, i.e. by adjusting the side of primer sequence
Formula improves primer sequence.Preferably, primer sequence and concentration are shown in Table 2.
2 primer sequence of table and corresponding concentration
The adjustment of embodiment 4PCR amplification system
In addition to the specificity amplification primer of said gene seat, this kit reaction system further includes that reaction mixture, heat open
Dynamic Taq enzyme and sdH2O, the reaction mixture include Tris-HCl, MgCl2, KCl, dNTPs, BSA, the concentration of each component can be with
It is adjusted as needed.
Specifically, in PCR amplification system, to Mg2+Two concentration, dNTPs concentration factor design orthogonal experiments carry out excellent
Change.Wherein, Mg2+Concentration tests 4 levels of 1.5mM, 2.0mM, 2.5mM, 3.0mM respectively, and dNTPs concentration is tested respectively
4 levels of 0.15mM, 0.2mM, 0.25mM, 0.30mM, Tris-HCl concentration are set to 50mM, and KCl concentration is set to 50mM.Finally
By Mg2+Concentration is set to 2.0mM, and dNTPs concentration is set to 0.2mM, and Tris-HCl concentration is set to 50mM, and KCl concentration is set to 50mM, benefit
It is prepared into PCR reaction mixture with the above material, is added in PCR system.Final PCR system each group is at being shown in Table 3.
3 PCR of table reacts amplification system
The application method of the kit of 5 the application of embodiment
1) debugging of amplification program: the kit based on the application needs to meet the adaptability and different annealing of different samples
The tolerance of temperature, it is therefore desirable to test different amplification programs, guarantee that different samples can obtain in wider temperature range
To correct DNA typing.First, it is preferable that recurring number may include 28 circulations, and 29 circulations, 30 circulations, 31 are followed
Ring, one of 32 circulations or any combination thereof；It is further preferable that recurring number can be 30 circulations.Secondly, annealing temperature
It may include 56 DEG C, 58 DEG C, 60 DEG C, 62 DEG C, one of 64 DEG C or any combination thereof；It is further preferable that temperature can be 60
℃.Through analyzing, most preferably, the amplification program of the kit of the application is shown in Table 4.
4 PCR amplification program of table
2) PCR amplification is carried out based on specific primer of the amplification program to the kit, comprising: table can be based on
The reaction amplification system of PCR shown in 3 is expanded on thermal cycler according to PCR response procedures shown in table 4, is obtained amplification and is produced
Object；
3) by the amplified production 3000rpm, it is centrifuged 5min, takes 1 μ L amplified production, 1 μ LAllelic Ladder respectively
It is mixed with 0.5 μ L AGCU Marker SIZ-600 and 12 μ L deionized formamides, ice bath after 95 DEG C of denaturation 3min passes through capillary
Electrophoresis tube detection, obtains genotyping result.
The application of the kit of 6 the application of embodiment
1) sample is collected, the sample is blood cake；
2) sample DNA extracts: being extracted using silica bead method in forensic DNA profiling laboratory inspection specification [GA-T383-2002]
Sample DNA；
3) PCR amplification and capillary electrophoresis detection are carried out using the kit of the application.Specific steps are as follows:
Firstly, carrying out fluorescent dye primer and PCR system preparation, PCR amplification and Capillary Electrophoresis inspection according to embodiment 1-5
Survey and finally obtain genotyping result, entire PCR amplification process time-consuming 2 hours or so.Testing result is shown in Fig. 3, Allelic
Ladder is shown in Fig. 2；
4) the Y chromosome STR kit (Yfiler Plus) and euchromosome STR kit produced using ABI company
(Identifiler) the sample DNA detection that step 2) is extracted, entire PCR amplification process time-consuming 5 hours or so are carried out respectively；
5) to the Y chromosome STR kit of ABI company production in the application kit of step 3) and step 4)
(Yfiler Plus) and euchromosome STR kit (Identifiler) carry out parting comparison, the results are shown in Table 5.
The kit of 5 the application of table is to huamn autosomal and Y chromosome genotyping result
As shown in Table 5,1) this kit realizes while detecting euchromosome STR locus and Y chromosome str locus
Seat, and genotyping result is accurate；2) it needs check problem to be twice reduced to primary completion originally, reduces cumbersome operation step
Suddenly, to reduce detection time, the chance of crime suspect is increased.
Several preferred embodiments of the invention have shown and described in above description, but as previously described, it should be understood that the present invention
Be not limited to forms disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations,
Modification and environment, and the above teachings or related fields of technology or knowledge can be passed through within that scope of the inventive concept describe herein
It is modified.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be in this hair
In the protection scope of bright appended claims.
Sequence table
<110>Anhui Public Security Bureau's material evidence evaluating center
Anhui peace section bioengineering (group) limited liability company
Zhongde Meilian Biotech Co., Ltd. Wuxi
<120>a kind of composite amplification reagent kit for detecting autosome and Y chromosome str locus seat simultaneously
<141> 2019-08-23
<160> 78
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tcttgtgtat ctattcattc aatcata 27
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gataggtagg caggcagata ggca 24
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtgcctttgg gggcatctct tat 23
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gacagagcaa gaccctgtct c 21
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gacagagcaa gaccctgtct c 21
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gggatgggtt gctggacatg gt 22
<210> 7
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gagacggggt ttcaccatgt tg 22
<210> 8
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gaattgcacc aaatattggt a 21
<210> 9
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
caaacaaagg cagatcccaa gc 22
<210> 10
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ttaaaaacct aatgacacag 20
<210> 11
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tgagccgaga tcacgccatt g 21
<210> 12
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
agttaatact ggacattgtg g 21
<210> 13
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
atatctatag atccatagat at 22
<210> 14
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gcacacacct gtaatcccag ctac 24
<210> 15
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
aagaatatcc tagctgtgaa tctc 24
<210> 16
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
aaggcacgtc ttacatgctg gcag 24
<210> 17
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
tggtctgttg tgggaccttg tgata 25
<210> 18
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
cagaactaat ggaatatcta tctat 25
<210> 19
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
agcacccaga accgtcgact g 21
<210> 20
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
tcccgctagg cccttctgtc 20
<210> 21
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
cccacacagt cccctgtac 19
<210> 22
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
ctagcagcag ctcatggtg 19
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
ggtgcctaaa gacttcatgg 20
<210> 24
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
atgagttatt cagtaagtta aag 23
<210> 25
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
atacagaatg gcactcttat t 21
<210> 26
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
tggcttccct tgttctgagg ct 22
<210> 27
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
caatccagca tgggtgacag agc 23
<210> 28
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gttggttaat tggaagccaa caat 24
<210> 29
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
aaaataaatg ctcatactga taga 24
<210> 30
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cccagcgctt tgggaggccg aggct 25
<210> 31
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
caacaacaag gatatagagt tag 23
<210> 32
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
tcctagttac tacctgagtg gagt 24
<210> 33
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
tatgggcgtg agtgcatgcc cat 23
<210> 34
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
atcattcaca gatgatagat gatagata 28
<210> 35
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
agctataatt gtaccactgc actcca 26
<210> 36
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ttggtgcacc cattacccga 20
<210> 37
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
taggttagat agagatagga 20
<210> 38
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
acaggtctag aggatccaag 20
<210> 39
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
gtatgagatc aaattgactg 20
<210> 40
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
ggcaccagga gtaatacttc 20
<210> 41
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
tggtcaggag cacacactcc a 21
<210> 42
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
cctccaggtt cccacccaac 20
<210> 43
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
acttgcaggg ctgaggcagg a 21
<210> 44
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
acaacacaaa taaacaaacc gtc 23
<210> 45
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
tgaactcaca gattaaactg taacca 26
<210> 46
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
atgactttgc gcttcaggac t 21
<210> 47
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
atgccctggg ctctgtaaag 20
<210> 48
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
atggcctggg ctctgtaaag 20
<210> 49
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
cacaggcttg aggccaacca 20
<210> 50
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
tatgtatttt tgtatttcat gtgta 25
<210> 51
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
ttaatttatt tacctatcct gt 22
<210> 52
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
aatagcaagt atgtgacaag g 21
<210> 53
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
gctttttagc caagtgattc 20
<210> 54
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
gtctccataa atatgtgagt ca 22
<210> 55
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
gatataactt gaaattaaac t 21
<210> 56
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
tagtaaggct tctccagaga g 21
<210> 57
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
ggataattac accatcagtt tc 22
<210> 58
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
aggtcgaagc tgaagtgag 19
<210> 59
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
agatatgtga ttagaagtac t 21
<210> 60
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
atttggcagc catcatggca gctgcc 26
<210> 61
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
tgccattctc ctgcttcagc ctc 23
<210> 62
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
atcttctacc tatcatcttt ctagctagct a 31
<210> 63
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
gatcagaatt ttggagaagg caca 24
<210> 64
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
gttttatttg tcattcctaa tgtggt 26
<210> 65
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
aaatgaggta tgtctcatag aaaagacat 29
<210> 66
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
gtcatagata gatgatggac tgcta 25
<210> 67
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
atctatctat cattatacct act 23
<210> 68
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
tatctatcta gcattatacc tact 24
<210> 69
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
ttaggtctaa catttaagtc tt 22
<210> 70
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
ggattacagg cataatccac catg 24
<210> 71
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
cacttcgtat caataagaat 20
<210> 72
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
tatttaccat atgttcatcc a 21
<210> 73
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
cctaagcaga gatgttggtt ttctg 25
<210> 74
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
atggtcaaaa caccatttcc 20
<210> 75
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
gggatgggaa atgatgtttc tgaa 24
<210> 76
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 76
aaacattgta ccagtaagtg cacg 24
<210> 77
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 77
tctggggttg ctttctgcta ggt 23
<210> 78
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 78
cttggtttgg ccataggata atga 24
Claims (13)
1. a kind of composite amplification reagent kit for detecting autosome and Y chromosome str locus seat simultaneously, which is characterized in that at least
Specificity amplification primer pair including 40 locus, sequence difference is successively are as follows: NO:1~2 SEQ ID, SEQ ID NO:3
~4, NO:5~6 SEQ ID, NO:7~8 SEQ ID, NO:9~10 SEQ ID, NO:11~12 SEQ ID, SEQ ID NO:
13~14, NO:15~16 SEQ ID, NO:17~18 SEQ ID, NO:19~20 SEQ ID, NO:21~22 SEQ ID,
NO:23~24 SEQ ID, NO:25~26 SEQ ID, NO:27~28 SEQ ID, NO:29~30 SEQ ID, SEQ ID
NO:31~32, NO:33~34 SEQ ID, NO:35~36 SEQ ID, NO:37~38 SEQ ID, SEQ ID NO:39~
40, NO:41~42 SEQ ID, NO:43~44 SEQ ID, NO:45~46 SEQ ID, NO:47~49 SEQ ID, SEQ ID
NO:50~51, NO:52~53 SEQ ID, NO:54~55 SEQ ID, NO:56~57 SEQ ID, SEQ ID NO:58~
59, NO:60~61 SEQ ID, NO:62~63 SEQ ID, NO:64~65 SEQ ID, NO:66~67 SEQ ID, SEQ ID
NO:66~68, NO:69~70 SEQ ID, NO:71~72 SEQ ID, NO:73~74 SEQ ID, SEQ ID NO:75~
76, NO:77~78 SEQ ID.
2. a kind of composite amplification reagent kit according to claim 1, which is characterized in that the specificity amplification primer is corresponding
40 str locus seats, 40 str locus seats are located at least one autosome and at least one Y chromosome, the base
Successively because of seat are as follows: DYS391, D3S1358, D13S317, D7S820, D16S539, Penta E, DYS635, DYS576,
DYS456、TPOX、TH01、D2S1338、D6S1043、DYS385a/b、DYS438、DYS448、DYS437、D19S433、vWA、
DYS19、CSF1PO、D18S51、FGA、Amelogenin、D8S1179、D5S818、D21S11、D12S391、Penta D、
DYS458, DYS533, DYS393, DYS389I/II, DYS439, DYS392, Y_GATA_H4, DYS390 and DYS481.
3. a kind of composite amplification reagent kit according to claim 1, which is characterized in that the end of the specificity amplification primer
Concentration is 0.15 μM~0.3 μM.
4. a kind of composite amplification reagent kit according to claim 2, which is characterized in that 40 locus to be grouped.
5. a kind of composite amplification reagent kit according to claim 4, which is characterized in that at least divide 40 locus
It is first group for 5 groups: DYS391, D3S1358, D13S317, D7S820, D16S539, Penta E, DYS635, DYS576；
DYS456, TPOX, TH01, D2S1338, D6S1043, DYS385a/b, DYS438, DYS448 are second group；DYS437,
D19S433, vWA, DYS19, CSF1PO, D18S51, FGA are third group；Amelogenin,D8S1179,D5S818,D21S11,
D12S391, Penta D, DYS458, DYS533 are the 4th group；DYS393,DYS389I/II,DYS439,DYS392,Y_GATA_
H4, DYS390, DYS481 are the 5th group.
6. a kind of composite amplification reagent kit according to claim 1, which is characterized in that will be in the specificity amplification primer
At least one primer 5 ' end be marked.
7. a kind of composite amplification reagent kit according to claim 6, which is characterized in that the corresponding spy of each group locus
5 ' ends of specific amplification primers use fluorochrome label.
8. a kind of composite amplification reagent kit according to claim 7, which is characterized in that the fluorescent dye include 6-FAM,
HEX, SUM, LYN, and/or PUR.
9. a kind of composite amplification reagent kit according to claim 4, which is characterized in that the kit includes reaction mixing
Liquid, specific composite amplification primer, hot start Taq polymerase, DNA standard items, sdH2O, Allelic Ladder, in fluorescent molecule amount
It marks, wherein the composition of reaction mixture are as follows: Tris-HCl 50mM, MgCl2 2.0mM、KCl 50mM、dNTPs 0.2mM、BSA
0.8mg/ml。
10. a kind of composite amplification reagent kit according to claim 9, which is characterized in that the amplification system of the kit
For reaction mixture: 10.0 μ L；Hot start Taq polymerase: 1.0 μ L；Primer mixture: 5.0 μ L；Genomic DNA: 0.125ng~
5.0ng；sdH2O complements to 25.0 μ L.
11. it is a kind of according to claim 1~any one of 10 described in composite amplification reagent kit application method, feature exists
In, when the specificity amplification primer is for when expanding, amplification program are as follows: 95 DEG C of 2min of denaturation recycle 94 DEG C of 30s, and 60 DEG C
40s, 72 DEG C of 1min 15 circulations, 90 DEG C of 30s, 58 DEG C of 1min, 65 DEG C of 1min20s 15 circulations terminate and extend 60 DEG C
20min, 4 DEG C of heat preservations maintain.
12. it is a kind of according to claim 1~any one of 10 described in composite amplification reagent kit in legal medical expert's individual identification, suspicion
Other is the application in investigation, and/or paternity identification.
13. application according to claim 12, which is characterized in that the composite amplification reagent kit is suitable for a plurality of types of
Sample, the people that the sample is extracted including the use of any one method in Chelex method, magnetic bead extraction method or organic method extraction process
Genomic DNA, and/or by filter paper, FTA card, cotton swab, gauze any one carrier collect human blood or oral cavity it is thin
Born of the same parents.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910784935.6A CN110423802A (en) | 2019-08-23 | 2019-08-23 | Composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910784935.6A CN110423802A (en) | 2019-08-23 | 2019-08-23 | Composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat |
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111286548A (en) * | 2020-04-13 | 2020-06-16 | 公安部物证鉴定中心 | Kit for detecting 68 loci based on next-generation sequencing technology and primer combination used by kit |
| CN112746096A (en) * | 2020-12-31 | 2021-05-04 | 郑州高新生物技术有限公司 | Human Y-STR detection method based on next-generation sequencing and application thereof |
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|---|---|---|---|---|
| CN105018623A (en) * | 2015-08-06 | 2015-11-04 | 无锡中德美联生物技术有限公司 | Fluorescence labeling combination amplification reagent kit capable of synchronously amplifying autosome gene loca and Y chromosome STR gene loca of people and application of fluorescence labeling combination amplification reagent kit |
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| CN111286548A (en) * | 2020-04-13 | 2020-06-16 | 公安部物证鉴定中心 | Kit for detecting 68 loci based on next-generation sequencing technology and primer combination used by kit |
| CN111286548B (en) * | 2020-04-13 | 2022-06-21 | 公安部物证鉴定中心 | Kit for detecting 68 loci based on next-generation sequencing technology and primer combination used by kit |
| CN112746096A (en) * | 2020-12-31 | 2021-05-04 | 郑州高新生物技术有限公司 | Human Y-STR detection method based on next-generation sequencing and application thereof |
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Application publication date: 20191108 |