CN107254516A - A kind of six color fluorescence STR classifying methods and system - Google Patents
A kind of six color fluorescence STR classifying methods and system Download PDFInfo
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Abstract
The present invention provides a kind of six colors fluorescence STR classifying methods and system, this method includes obtaining individual DNA, obtain individual DNA genotype of totally 25 locus including 24 euchromosome STR locus and 1 Sex Determination locus Amelogenin, 24 euchromosome STR locus are D5S818, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, vWA, D8S1179, D16S539, Penta E, TPOX, TH01, D19S433, D18S51, FGA, D6S1043, D13S317, D12S391, Penta D, D2S441, D1S1656, D22S1045, D10S1248 and D11S4463, and the individual STR genotyping results are obtained according to the genotype of 25 locus.The present invention program breaks through limitation of the 5 color fluorescence to composite amplification locus quantity, makes to detect more efficient to DNA, graph-spectrum quality is higher, is as a result easy to analysis, and adapt to newest capillary electrophoresis detection Platform Requirements.
Description
Technical field
The present invention relates to a kind of classifying method and system, more particularly to a kind of six colors fluorescence STR classifying methods and system.
Background technology
Current legal medical expert STR detections using ripe florescence labeling STR multiplex kit, mainly for case and
New demand of both DNA database establishments is studied.For case angle, the difficulty that current public security organ DNA handles a case
Point is how to use as far as possible few more correct hereditary information of sample acquisition, in some particular cases (such as：Rape, gang-rape case)
And paternity identification is (such as：Father and daughter, sister's Relationship iden- tification) genetic markers such as Y-STR, X-STR are also carried out in case；It is another
Aspect is with the continuous expansion of China's DNA database sizes, to help more clearly to differentiate the matched data being retrieved, and compels
More str locus seats will be introduced by being essential.In special campaigns of cracking down on the abduction, due to the presence of str locus seat jumping phenomenon, usually need
More str locus seat is examined just to make judgement definitely.Therefore, integrated in same composite amplification system more
Ground str locus seat is the developing direction of current main flow.In addition, improve DNA detection reagents anti-rejection ability and system it is sensitive
Degree, simplified operating procedure, the detection time that shortens, raising detection efficiency are also the important requirement to the system.
The kit of current China independent research is still based on multicolored fluorescence, by Recent study, due to amplification target
Fragment length is limited, and the locus number that each color can be accommodated is limited, and fluorescent species are to determine that kit can detect base
One of key factor because of seat number, multicolored fluorescence turns into restriction, and we improve the big bottleneck that kit detects efficiency.Although
Some import reagent boxes can detect six color fluorescence, but its is expensive, and due to different geographical, national locus polymorphic
Difference is huge, and import reagent box can not largely meet the STR partings requirement for Chinese population.
The six color fluorescence STR classifying methods and system for Chinese population are how developed, case analysis requirement is met, improved
Detection efficiency, which turns into, technical problem to be solved.
The content of the invention
The invention provides a kind of six colors fluorescence STR classifying methods, combine and be directed to by using specific str locus seat
The specific primer and fluorescence labeling of these locus design, can be to the normal sample of Chinese population individual and micro degraded sample
Precise and high efficiency parting is carried out, so as to provide data support to carry out individual identification or paternity identification etc..
Present invention also offers a kind of six colors fluorescence STR classification systems, it can be realized to for 25 bases by the system
Because of the accurate parting of seat.
Present invention also offers a kind of compound detection system, the detection architecture can accurately obtain 25 str locus
Genotype.
Present invention also offers a kind of detection kit, including described compound detection system.
A kind of six colors fluorescence STR classifying methods that the present invention is provided, including：
1) individual DNA is obtained；
2) obtaining the individual DNA includes 24 euchromosome STR locus and 1 Sex Determination locus
The genotype of totally 25 locus including Amelogenin, 24 euchromosome STR locus are D5S818, D21S11,
D7S820,CSF1PO,D2S1338,D3S1358,vWA,D8S1179,D16S539,Penta E,TPOX,TH01,D19S433,
D18S51, FGA, D6S1043, D13S317, D12S391, Penta D, D2S441, D1S1656, D22S1045, D10S1248 and
D11S4463, including use and it is expanded with the one-to-one amplimer of 25 locus to obtain amplified production
The step of,
The fluorescence labeling of the corresponding amplimer of each locus is：Amelogenin, D5S818, D21S11,
The fluorescence labeling of D7S820, CSF1PO and D2S1338 amplimer is FAM；D3S1358, vWA, D8S1179, D16S539 and
The fluorescence labeling of Penta E amplimer is HEX；TPOX, TH01, D19S433, D18S51 and FGA amplimer it is glimmering
Cursor is designated as TAMRA；The fluorescence labeling of D6S1043, D13S317, D12S39 and Penta D amplimer is ROX；
The fluorescence labeling of D2S441, D1S1656, D22S1045, D10S1248 and D11S4463 amplimer is SYP；
3) the individual STR genotyping results are obtained according to the genotype of 25 locus.
In the solution of the present invention, 25 locus be applicant by the living environment to a large amount of Chinese populations,
Ethnic origin etc. carries out comprehensive analysis acquisition.Primer for said gene seat is also that applicant is obtained by many experiments
, the combination of these specific genes seat is realized to normal sample and micro degraded sample (such as：Contact DNA, cast-off cells are rotten
Lose sample etc.) DNA typing.
Further, in the scheme of the application, " such as blood, group that individual DNA " refers to extracting the individual are obtained
The DNA in equal samples is knitted, or directly obtains the blood card containing the individual DNA." the individual STR genotyping results " refers to
It is that can be used for the STR genotyping results to the carry out individual identification or paternity identification.
In the specific embodiment of the present invention, the amplimer is SEQ ID No.1 to SEQ in sequence table
ID No.50 nucleotide sequence.
In the another embodiment of the present invention, wherein 2) being additionally included in after acquisition amplified production, heredity is used
Analyzer analyzes the amplified production, the step of genotype to obtain 25 locus.It is described in the solution of the present invention
Genetic analyzer can be the conventional use of genetic analyzer of those skilled in the art, such as ABI3130 or ABI3500 types heredity
Analyzer, passes throughID-X softwares or other GeneMapper softwares etc. analyze institute in the pcr amplification product
State the genotype of 25 locus.
A kind of six colors fluorescence STR classification systems that the present invention is provided, including DNA obtain system, compound detection system, deduction
System；
The DNA, which obtains system, to be used to obtain individual DNA；
The compound detection system includes 24 euchromosome STR locus and 1 sex for obtaining the individual DNA
The genotype of totally 25 locus is determined including locus Amelogenin, 24 euchromosome STR locus are
D1S1656,D2S1338,D2S441,D3S1358,D5S818,D6S1043,D7S820,D8S1179,D10S1248,
D12S391,D13S317,D16S539,D18S51,D19S433,D21S11,D22S1045,CSF1PO,FGA,Penta D,
Penta E, TH01, TPOX, vWA and D11S4463, including use and the one-to-one amplimer pair of 25 locus
The step of it is expanded to obtain amplified production；
The fluorescence labeling of the corresponding amplimer of each locus is：Amelogenin, D5S818, D21S11,
The fluorescence labeling of D7S820, CSF1PO and D2S1338 amplimer is FAM；D3S1358, vWA, D8S1179, D16S539 and
The fluorescence labeling of Penta E amplimer is HEX；TPOX, TH01, D19S433, D18S51 and FGA amplimer it is glimmering
Cursor is designated as TAMRA；The fluorescence labeling of D6S1043, D13S317, D12S39 and Penta D amplimer is ROX；
The fluorescence labeling of D2S441, D1S1656, D22S1045, D10S1248 and D11S4463 amplimer is SYP；
The deduction system is used for according to the individual STR genotyping results to the carry out individual identification or paternity identification.
In the specific embodiment of the present invention, the amplimer is SEQ ID No.1 to SEQ in sequence table
ID No.50 nucleotide sequence.
Further, the compound detection system is additionally operable to after amplified production is obtained, and is analyzed using genetic analyzer
The amplified production, to obtain the genotype of 25 locus.
Further, the genetic analyzer utilizes the parting standard thing for the allele of each locus to determine
The genotype of each locus in DNA.The preparation of the parting standard thing of allele can be prepared according to this area conventional meanses.
A kind of compound detection system that the present invention is provided, the system includes individual DNA, 25 locus, and amplification
Primer,
The compound detection system includes 24 euchromosome STR locus and 1 sex for obtaining the individual DNA
The genotype of totally 25 locus is determined including locus Amelogenin, 24 euchromosome STR locus are
D1S1656,D2S1338,D2S441,D3S1358,D5S818,D6S1043,D7S820,D8S1179,D10S1248,
D12S391,D13S317,D16S539,D18S51,D19S433,D21S11,D22S1045,CSF1PO,FGA,Penta D,
Penta E, TH01, TPOX, vWA and D11S4463, including use and the one-to-one amplimer pair of 25 locus
The step of it is expanded to obtain amplified production, the amplimer is SEQ ID No.1 to SEQ ID in sequence table
No.50 nucleotide sequence.
Further, the fluorescence labeling of the corresponding amplimer of each locus is in the system：Amelogenin,
The fluorescence labeling of D5S818, D21S11, D7S820, CSF1PO and D2S1338 amplimer is FAM；D3S1358,vWA,
The fluorescence labeling of D8S1179, D16S539 and Penta E amplimer is HEX；TPOX, TH01, D19S433, D18S51 and
The fluorescence labeling of FGA amplimer is TAMRA；D6S1043, D13S317, D12S39 and Penta D amplimer it is glimmering
Cursor is designated as ROX；The fluorescence labeling of D2S441, D1S1656, D22S1045, D10S1248 and D11S4463 amplimer is
SYP。
In the solution of the present invention, the archaeal dna polymerase can be Fast Start archaeal dna polymerases, Taq DNA polymerizations
One or more in enzyme, Hotstart archaeal dna polymerases.
Present invention also offers a kind of detection kit, including described compound detection system.
In the solution of the present invention, the present invention carries out the side of the parting of 25 locus using the compound detection system
Method, including：1) it regard the DNA of acquisition as template；2) amplimer is used to carrying out multiplex PCR expansion as the DNA of template
Increasing is reacted to give amplified production；3) amplified production is analyzed using genetic analyzer, to obtain 25 locus
Genotype.
In the solution of the present invention, the 25 locus information is as shown in table 1：
Table 1
The amplimer sequence that the present invention is provided is as follows.The amplimer and its corresponding locus such as table 2 below institute
Show, PCRU represents sense primer, PCRL represents anti-sense primer；
Table 2
The present invention program has the following advantages that：
1st, 6 color fluorescence STR classifying methods of the invention and classification system are using the combination of specific str locus seat and primer
Sequence so that arranged on the parting collection of illustrative plates finally obtained between each site uniform, collection of illustrative plates is completely clear；Testing result is repeated without bright
Significant difference is different；Stutter peak area ratios are no more than 10%, and ratio of peak is no more than 15%, are fully able to meet wanting for legal medical expert STR inspections
Ask.
2nd, specific 25 STR bits can be detected simultaneously using the 6 color fluorescence STR classifying methods and classification system of the present invention
Point, with high individual identification or paternity identification ability.Limitation of the 5 color fluorescence to composite amplification locus quantity is breached, is made
The use detected to DNA is more efficient, and graph-spectrum quality is higher, is as a result easy to analysis.
3rd, the solution of the present invention can be using conventional genetic analyzer, according to the molecular weight and fluorescence of the gene to be expanded
The difference of color, obtains intuitively testing result, and the result, compared with existing detecting system, accuracy is 100%.
4th, the solution of the present invention detection sensitivity reaches 0.125ng, and system is reproducible, and parting is accurate, and can fit
The requirement of newest capillary electrophoresis detection platform is answered, the accumulative matching probability (Pm) having is 3.5434 × 10-28, add up individual
Discernment (TDP) is 0.999 999 999 999 999 999 999999969863, adds up non-father's probability of exclusion (CEP) and is
0.999 99999 375。
Brief description of the drawings
Fig. 1 shows the genotyping result figure of the sample obtained using present system.
Fig. 2 shows the sensitivity technique result of the inventive method and system.
Fig. 3 shows the species specificity testing result of the inventive method and system.
Fig. 4 shows the repeated testing result of the inventive method and system.
Embodiment
The 180 parts of unrelated person blood cards (90 parts of male, 90 parts of women) used in following examples, by the Ministry of Public Security, material evidence reflects
Center offer.
The method used in following examples is conventional method unless otherwise instructed, and agents useful for same consumptive material and instrument are as follows
Shown in table 3：
Table 3
| Tris-HCl | Sigma-Aldrich |
| EDTA | Sigma-Aldrich |
| MgCl2 | Sigma-Aldrich |
| KCl | Sigma-Aldrich |
| dNTP | Amresco companies of the U.S. |
| NaCl | Chemical Reagent Co., Ltd., Sinopharm Group |
| Absolute ethyl alcohol | Chemical Reagent Co., Ltd., Sinopharm Group |
| Hot start Taq polymerase | Roche companies of Switzerland |
| Typer500 internal standards | Material Evidence Identification Center, Ministry of Public Security |
| Deionized formamide | Applied Biosystems companies of the U.S. |
| Capillary electrophoresis buffer | Applied Biosystems companies |
| ABI 3500XL type genetic analyzers | Applied Biosystems |
| Proflex PCR amplification instruments | Applied Biosystems |
Embodiment 1, the checking to the method and system accuracy to carrying out STR partings of the invention
In the present embodiment, described is 180 parts of human blood cards, it is known that its individual source, but in the implementation of the embodiment of the present application 1
During set its individual source it is unknown, STR partings are carried out to it using the application method and system, including：
1) it is 180 parts of human blood cards, 2 to obtain system to obtain the individual DNA using the DNA in the system of the present invention)
Using the genotype of the individual 25 str locus seats of DNA of the compound detection system acquisition in the system of the present invention, and according to
The genotype obtains the individual STR genotyping results, 3) infer system using described in system of the invention, according to the individual
STR genotyping results to the carry out individual identification or paternity identification.
In the present embodiment, the compound detection system includes 24 euchromosome STR genes for obtaining the individual DNA
The genotype of totally 25 locus, 24 euchromosome STR bases including seat and 1 Sex Determination locus Amelogenin
Because seat be D1S1656, D2S1338, D2S441, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248,
D12S391,D13S317,D16S539,D18S51,D19S433,D21S11,D22S1045,CSF1PO,FGA,Penta D,
Penta E, TH01, TPOX, vWA and D11S4463, including use and the one-to-one amplimer pair of 25 locus
The step of it is expanded to obtain amplified production, the amplimer is SEQ ID No.1 to SEQ ID in sequence table
No.50 nucleotide sequence；
The fluorescence labeling of the corresponding amplimer of each locus is：Amelogenin, D5S818, D21S11,
The fluorescence labeling of D7S820, CSF1PO and D2S1338 amplimer is FAM；D3S1358, vWA, D8S1179, D16S539 and
The fluorescence labeling of Penta E amplimer is HEX；TPOX, TH01, D19S433, D18S51 and FGA amplimer it is glimmering
Cursor is designated as TAMRA；The fluorescence labeling of D6S1043, D13S317, D12S39 and Penta D amplimer is ROX；
The fluorescence labeling of D2S441, D1S1656, D22S1045, D10S1248 and D11S4463 amplimer is SYP, in the present invention
Above-mentioned amplimer is dyed using normal dyeing means in scheme.
1st, template：180 parts of human blood cards.
2nd, the parting of 25 locus is carried out to above-mentioned template using the compound detection system, including：Expanded using described
Increase primer and carry out multiplexed PCR amplification, to obtain amplified production；Amplified production is determined into 25 locus using genetic analyzer
Genotype.Detailed process is as follows：
2.1st, primer pond is configured
The configuration in amplimer pond, wherein the one-to-one 25 pairs of amplimers of 25 locus are as described above；This
The various primer sequences that invention is provided are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Synthetic primer is diluted to 100 μM with 1 × TE buffer solutions, by upstream and downstream primer of 25 locus etc. than mixed
Close, obtain the primer that concentration is 50 μM.Different volumes are taken to be added in a new centrifuge tube respectively from 25 pipe PCR primers,
As 25 heavy PCR primer ponds, in the primer pond, final concentration of 0.1-0.3 μM of the primer of each STR bit point.
2.2nd, multi-PRC reaction
The present embodiment carries out multi-PRC reaction using Proflex PCR amplification instruments.
(1) configuration PCR reactant mixtures (PCR mix) (10 μ L systems), as shown in table 4 below.
Table 4
(2) amplification program
PCR reactant mixtures are placed in PCR instrument, adjustment cyclic program such as table 5 below is expanded, and obtains amplified production：
Table 5
2.3rd, PCR primer parting
Treat the preparation of parting sample：
1. the preparation of electrophoresis loading mixture, is prepared to constitute loading mixing by internal standard and deionized formamide in following ratio
Thing：The μ l deionized formamides of 10 μ l Typer500 internal standards+1000, are well mixed.
2. often pipe adds 10 μ l loadings mixtures, 1 μ l amplified productions, mix.
3.95 DEG C be denatured 3 minutes, be immediately placed at cooled on ice 3 minutes, rear electrophoresis.
Using being detected on ABI 3500XL type genetic analyzers.Using ABI 3500XL Date Collection
Software 3.1 collects data, and Genemapper IDX v1.4 softwares are analyzed electrophoresis result, obtains 25 bases
Because of the genotype of seat.
2.4th, interpretation of result
180 parts of DNA samples are carried out with a representative genotyping result of parting as shown in figure 1, being using present system
The accuracy of checking genotyping result, randomly selects 100 parts of DNA samples from 180 parts of DNA samples, and 25 locus are carried out
It is sequenced (sequencing of Beijing Mai Aodeen bio tech ltd), described obtained using the compound detection system of the present embodiment
All genotyping results of body are consistent with sequencing result, and uniformity reaches 100%, and this result proves compound detection system of the present invention
Genotyping result is accurate.
3rd, individual identification is carried out to it according to the genotype of unknown source individual DNA 25 locus
The individual source results of the above-mentioned 180 parts of samples obtained by the present embodiment method, are tied with its known individual source
Fruit is consistent, illustrates that the inventive method can carry out individual identification to unknown source individual.
4th, according to 180 parts of DNA samples 25 locus genotype, obtain the STR genotyping results of individual, and carry out
Paternity identification.The above-mentioned 180 individual paternity identification results obtained by the present embodiment method, reflect with its known individual parental right
Determine result consistent, illustrate the paternity identification that the inventive method can be carried out.
The composite amplification system sensitivity of the present invention of embodiment 2, species specificity, system repeatability, genotyping result accuracy
Checking
First, sensitivity technique
As 9947 standard DNA template amounts >=0.125ng, whole allele of 25 locus can correct parting, see
Fig. 2.Template amount<, there is allelic loss phenomenon in 0.125ng.I.e. system of the invention can be entered to as little as 0.125ng DNA
Row detection.
2nd, species specificity is detected
Composite amplification system detects animal (pig, dog, sheep, rabbit and Escherichia coli) blood sample, is not produced in parting line zone
Specific peak type, is shown in Fig. 3.It can be seen that the method and system of the present invention has good species specificity.
3rd, system repeatability detection
100 parts of samples (venous blood, from Material Evidence Identification Center, Ministry of Public Security) are detected using the reagent of 2 batches,
As a result stable, collection of illustrative plates is completely clear；Repeat testing result no significant difference；Stutter peak area ratios are no more than 10%, ratio of peak
No more than 15%.Using system parting of the present invention with usingPlus PCR
Amplification Kit kits partings genotyping result on shared locus is identical, illustrate the method for the present invention with
System has good system repeatability.
Further, by PCR primer+reactant mixture in PCR system of the present invention and Typer500 internal standards multigelation 5,
10th, 15, there is no to expand balance between the testing result that 20 times obtain between significant change, each locus good, referring to Fig. 4 (repeatedly
20 results of freeze thawing), it can be seen that method and system of the invention has good system repeatability.
4th, all kinds of case sample parting accuracys
All kinds of common samples such as blood stain sample, bone and the salivary stain equal samples of storage 15 years, 25 through present system
Individual str locus seat composite amplification system detection can obtain accurate genotyping result, and peak type is sharp, and amplification balance is good, and with
DNATyper 19TM、Plus PCR Amplification Kit kits are shared
Genotyping result on locus is identical.
Genetic parameters (n=209) of the 7 24 str locus seats of table in Chinese han population
Using the data in above table, the accumulative matching probability of application, cumulative individual are obtained according to below equation and recognized
Rate and accumulative non-father's probability of exclusion.The frequency of i-th phenotype in Pm (accumulative matching probability)=∑ pi2pi colonies.TDP
(cumulative individual discrimination)=1- (1-DP1)(1-DP2)(1-DP3)…(1-DPK), wherein DPKFor the DP values of k-th of locus.
CPE (adding up non-father's probability of exclusion)=1- (1-PE1) (1-PE2) (1-PE3) ... (1-PEK), wherein PEKFor k-th of locus
PE values.
24 str locus accumulative matching probability (Pm) for having in Chinese han population of seat of the present invention for 3.5434 ×
10-28, it is 0.999 999 999 999 999 999 9,999,999 69863 to add up individual identification power (TDP), adds up non-father row
Except probability (CEP) is 0.999 99,999 375.
Sequence table
<110>Material Evidence Identification Center, Ministry of Public Security
<120>A kind of six color fluorescence STR classifying methods and system
<130> 168727GF
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<213>Artificial sequence
<220>
<223>Primer
<400> 17
ttgcaactta tatgtatttt tgtatttc 28
<210> 18
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 18
ccaaattgtg ttcatgagta tagtt 25
<210> 19
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 19
gatcccaagc tcttcctctt 20
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 20
acgtttgtgt gtgcatctgt 20
<210> 21
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 21
taccaacatg aaagggtacc aa 22
<210> 22
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 22
ttattaattg agaaaactcc ttacaat 27
<210> 23
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 23
ctagcaccca gaaccgt 17
<210> 24
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 24
ttgtcagcgt ttatttg 17
<210> 25
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 25
ttccgagtgc aggtca 16
<210> 26
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 26
gctgccctag tcagc 15
<210> 27
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 27
ctgggcaaca gaataagat 19
<210> 28
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 28
aggtttttaa ggaacaggtg g 21
<210> 29
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 29
cttgagccca gaaggt 16
<210> 30
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 30
tctaccagca acaacacaaa ta 22
<210> 31
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 31
cataggtttt gaactc 16
<210> 32
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 32
ttgtctgtaa ttgccag 17
<210> 33
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 33
aaggatgggt ggatcaat 18
<210> 34
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 34
tgtatgagcc acttccca 18
<210> 35
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 35
cagaagtctg ggatgtgg 18
<210> 36
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 36
cccaaaaaga cagacag 17
<210> 37
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 37
acaggatcaa tggatgcat 19
<210> 38
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 38
tggcttttag acctggact 19
<210> 39
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 39
aaggtcgaag ctgaagt 17
<210> 40
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 40
tagaattctt taatctggac a 21
<210> 41
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 41
tgtggctcat ctatgaaaac t 21
<210> 42
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 42
gaagtggctg tggtgttatg 20
<210> 43
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 43
gtgttgctca agggtca 17
<210> 44
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 44
aaatagaatc actagggaac 20
<210> 45
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 45
ttccccgatg atagtagt 18
<210> 46
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 46
gcgaatgtat gattggcaat 20
<210> 47
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 47
aatgaattga acaaatgagt g 21
<210> 48
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 48
gcaactctgg ttgtattgtc t 21
<210> 49
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 49
atcctattgg aggccatca 19
<210> 50
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 50
gattgacctg tctgtccg 18
Claims (9)
1. a kind of six colors fluorescence STR classifying methods, it is characterised in that this method includes：
1) individual DNA is obtained；
2) the individual DNA is obtained including 24 euchromosome STR locus and 1 Sex Determination locus Amelogenin to exist
The interior genotype of totally 25 locus, 24 euchromosome STR locus are D5S818, D21S11, D7S820,
CSF1PO,D2S1338,D3S1358,vWA,D8S1179,D16S539,Penta E,TPOX,TH01,D19S433,D18S51,
FGA, D6S1043, D13S317, D12S391, Penta D, D2S441, D1S1656, D22S1045, D10S1248 and
D11S4463, including use and it is expanded with the one-to-one amplimer of 25 locus to obtain amplified production
The step of,
The fluorescence labeling of the corresponding amplimer of each locus is：Amelogenin, D5S818, D21S11, D7S820,
The fluorescence labeling of CSF1PO and D2S1338 amplimer is FAM；D3S1358, vWA, D8S1179, D16S539 and Penta E
Amplimer fluorescence labeling be HEX；The fluorescence labeling of TPOX, TH01, D19S433, D18S51 and FGA amplimer is
TAMRA；The fluorescence labeling of D6S1043, D13S317, D12S39 and Penta D amplimer is ROX；D2S441,
The fluorescence labeling of D1S1656, D22S1045, D10S1248 and D11S4463 amplimer is SYP；
3) the individual STR genotyping results are obtained according to the genotype of 25 locus.
2. according to the method described in claim 1, it is characterised in that the amplimer is that SEQ ID No.1 are extremely in sequence table
SEQ ID No.50 nucleotide sequence.
3. according to the method described in claim 1, it is characterised in that wherein 2) be additionally included in after acquisition amplified production, use something lost
Pass analyzer and analyze the amplified production, the step of genotype to obtain 25 locus.
4. a kind of six colors fluorescence STR classification systems, it is characterised in that the system includes DNA and obtains system, compound detection body
System, deduction system；
The DNA, which obtains system, to be used to obtain individual DNA；
The compound detection system includes 24 euchromosome STR locus and 1 Sex Determination for obtaining the individual DNA
The genotype of totally 25 locus including locus Amelogenin, 24 euchromosome STR locus are D1S1656,
D2S1338,D2S441,D3S1358,D5S818,D6S1043,D7S820,D8S1179,D10S1248,D12S391,
D13S317,D16S539,D18S51,D19S433,D21S11,D22S1045,CSF1PO,FGA,Penta D,Penta E,
TH01, TPOX, vWA and D11S4463, including use are expanded it with the one-to-one amplimer of 25 locus
The step of increasing to obtain amplified production；
The fluorescence labeling of the corresponding amplimer of each locus is：Amelogenin, D5S818, D21S11, D7S820,
The fluorescence labeling of CSF1PO and D2S1338 amplimer is FAM；D3S1358, vWA, D8S1179, D16S539 and Penta E
Amplimer fluorescence labeling be HEX；The fluorescence labeling of TPOX, TH01, D19S433, D18S51 and FGA amplimer is
TAMRA；The fluorescence labeling of D6S1043, D13S317, D12S39 and Penta D amplimer is ROX；D2S441,
The fluorescence labeling of D1S1656, D22S1045, D10S1248 and D11S4463 amplimer is SYP；
The deduction system is used for according to the individual STR genotyping results to the carry out individual identification or paternity identification.
5. system according to claim 4, it is characterised in that the amplimer is that SEQ ID No.1 are extremely in sequence table
SEQ ID No.50 nucleotide sequence.
6. system according to claim 5, it is characterised in that the compound detection system is additionally operable to obtaining amplified production
Afterwards, the amplified production is analyzed using genetic analyzer, to obtain the genotype of 25 locus.
7. a kind of compound detection system, it is characterised in that the system includes：Individual DNA, 25 locus, amplimer,
The compound detection system includes 24 euchromosome STR locus and 1 Sex Determination for obtaining the individual DNA
The genotype of totally 25 locus including locus Amelogenin, 24 euchromosome STR locus are D1S1656,
D2S1338,D2S441,D3S1358,D5S818,D6S1043,D7S820,D8S1179,D10S1248,D12S391,
D13S317,D16S539,D18S51,D19S433,D21S11,D22S1045,CSF1PO,FGA,Penta D,Penta E,
TH01, TPOX, vWA and D11S4463, including use are expanded it with the one-to-one amplimer of 25 locus
The step of increasing to obtain amplified production, the nucleosides that the amplimer is SEQ ID No.1 to SEQ ID No.50 in sequence table
Acid sequence.
8. compound detection system according to claim 7, it is characterised in that the corresponding amplification of each locus in the system
The fluorescence labeling of primer is：Amelogenin, D5S818, D21S11, D7S820, CSF1PO and D2S1338 amplimer
Fluorescence labeling is FAM；The fluorescence labeling of D3S1358, vWA, D8S1179, D16S539 and Penta E amplimer is HEX；
The fluorescence labeling of TPOX, TH01, D19S433, D18S51 and FGA amplimer is TAMRA；D6S1043,D13S317,
The fluorescence labeling of D12S39 and Penta D amplimer is ROX；D2S441, D1S1656, D22S1045, D10S1248 and
The fluorescence labeling of D11S4463 amplimer is SYP.
9. a kind of detection kit, it is characterised in that including the compound detection system described in claim 7 or 8.
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| CN111286320A (en) * | 2020-01-22 | 2020-06-16 | 公安部物证鉴定中心 | Fluorescent dye for nine-color fluorescent STR typing, specific amplification primer pair and typing method |
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| CN113684263B (en) * | 2021-09-26 | 2024-05-10 | 公安部第一研究所 | Eight-color fluorescent STR composite amplification detection kit and application thereof |
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