CN106480198A - A kind of method and system for carrying out individual identification to unknown sample - Google Patents
A kind of method and system for carrying out individual identification to unknown sample Download PDFInfo
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Abstract
The present invention provides a kind of method and system for carrying out individual identification to unknown sample，The method includes to extract unknown sample DNA，Obtain genotyping result of the DNA including totally 31 locus including 30 InDel locus and 1 sex identification locus Amelogenin，30 InDel locus are rs10629077、rs2308026、rs361519、rs2307652、rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、rs2307553、rs16438、rs2307981、rs4646006、Rs2308072 and rs140864；Genotype according to 31 locus of unknown sample carries out individual identification.The solution of the present invention can realize the individual identification to unknown sample in Chinese population using InDel locus.
Description
Technical field
The present invention relates to a kind of method and system of individual identification, more particularly to one kind carry out individual identification to unknown sample
Method and system.
Background technology
Insertion-deletion polymorphism (insertion-deletion, InDel) is the insertion of section of DNA fragment or lacks
Become homeless two polymorphic alleles of the specific type to be formed.There is following feature with respect to STR and SNP, InDel：(1) wide
General is distributed in whole gene group；(2) because of single mutational events, and occurrence frequency is low, more stable after generation, is difficult recurrence
Mutation；(3) first ancestor's informative site can be become, judges that region is originated；(4) InDel can be expanded in less amplicon, fitted
Detection for degradation of dna；(5) InDel can carry out Genotyping using the existing instrument and equipment of a forensic DNA laboratory；(6)
InDel goes for automation and high-throughout technology simultaneously.
Therefore, InDel is paid close attention to by Chinese scholars, and existing multidigit scholar is established suitable for many to this nationality
The compound system of state property research.But as in Chinese population, various nationalities' allele is various, how to establish a set of based on InDel
The locus, composite amplification system of the identification of Forensic DNA that is being applied to extensive Chinese population, makes replacement STR detection
Another powerful measure of system anlysis DNA sample, becoming has problem to be solved.
Content of the invention
The invention provides a kind of method for carrying out individual identification to unknown sample, can obtain unknown sample includes 30
InDel locus and 1 sex identification locus in the genotyping result of interior totally 31 locus, so as to realize to unknown sample
Individual identification.
The present invention also provides a kind of system for carrying out individual identification to unknown sample, can be realized to unknown by the system
Sample is directed to the accurate parting of above-mentioned 31 locus, so as to carry out individual identification to unknown sample.
Present invention also offers a kind of compound detection system, the detection architecture can accurately obtain unknown sample includes 30
InDel locus and 1 sex identification locus are in the genotyping result of interior totally 31 locus.
Present invention also offers a kind of detection kit, including described compound detection system.
A kind of method for carrying out individual identification to unknown sample that the present invention is provided, the method include：
1) DNA of unknown sample is extracted；
2) DNA is obtained including including 30 InDel locus and 1 sex identification locus Amelogenin totally 31
The genotyping result of individual locus, 30 InDel locus be rs10629077, rs2308026, rs361519,
rs2307652、rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、
rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、
rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、
Rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and rs140864；
3) individual identification is carried out according to the genotype of 31 locus of unknown sample.
In the solution of the present invention, 31 locus are applicants by living environment, the race to Chinese population
Origin etc. carries out comprehensive analysis, investigates the phenotypic characteristic difference of each department nationality population, including resemblance, physical signs etc.,
For these differences carry out document and network data base investigation, on the basis of having studied obtain can carry out individual identification and
The combination of the specific gene seat of paternity identification.The unknown sample can be from the blood sample of human body, cast-off cells, bone,
Tooth, seminal stain and buccal swab equal samples, the individuality source of these samples are unknown.
In a specific embodiment of the present invention, 2) are included using one-to-one with 31 locus
The step of 31 pairs of amplimers are expanded to which to obtain amplified production；The amplimer is SEQ ID in sequence table
The nucleotide sequence of No.1 to SEQ ID No.62.
In the another embodiment of the present invention, wherein 2) after being additionally included in acquisition amplified production, using heredity
The amplified production analyzed by analyzer, with obtain 31 locus genotype the step of.In the solution of the present invention, described
Genetic analyzer can be the conventional use of genetic analyzer of those skilled in the art, such as ABI3130 or ABI3500 type heredity
Analyzer, passes throughInstitute in the pcr amplification product analyzed by ID-X software or other GeneMapper softwares etc.
State the genotype of 31 locus.
A kind of system for carrying out individual identification to unknown sample that the present invention is provided, the system include that DNA extracts system,
Compound detection system, and infer system；The DNA extracts system is used for extracting the DNA of unknown sample；The compound detection body
Be for obtaining the DNA including totally 31 bases including 30 InDel locus and 1 sex identification locus Amelogenin
Because of the genotyping result of seat, 30 InDel locus are rs10629077, rs2308026, rs361519, rs2307652,
rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、rs1305056、
rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、rs2308020、
rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、rs2307553、
Rs16438, rs2307981, rs4646006, rs2308072 and rs140864；
The deduction system is used for carrying out individual identification according to the genotype of 31 locus of unknown sample.
In a specific embodiment of the present invention, the compound detection system is used for adopting and 31 locus
One-to-one 31 pairs of amplimers are expanded to which to obtain amplified production, and the amplimer is SEQ ID in sequence table
The nucleotide sequence of No.1 to SEQ ID No.62.
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 31 locus.
A kind of compound detection system that the present invention is provided, the system include unknown sample DNA, 31 locus, and
Amplimer, the compound detection system include 30 InDel locus and 1 sex identification gene for obtaining the DNA
Seat Amelogenin interior totally 31 locus genotyping result, 30 InDel locus be rs10629077,
rs2308026、rs361519、rs2307652、rs16671、rs33948716、rs8190507、rs1610937、
rs2307689、rs2307976、rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、
rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、
Rs3047269, rs2067294, rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and
rs140864；The amplimer by constituting with the one-to-one 31 pairs of amplimers of 31 locus, draw by the amplification
Thing is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in sequence table.
In the solution of the present invention, the archaeal dna polymerase can be Fast Start archaeal dna polymerase, Taq DNA polymerization
One or more in enzyme, Hotstart archaeal dna polymerase.
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 31 locus using the compound detection system
Method, including：1) using the unknown sample DNA for extracting as template；2) amplimer is used to the unknown sample as template
DNA carries out multiplexed PCR amplification and is reacted to give amplified production；3) amplified production is analyzed using genetic analyzer,
To obtain the genotyping result of 31 locus.
The present invention program has the following advantages that：
1st, the method for the present invention and system carry out individual identification using specific 31 locus, and these locus are in the Chinese
In the national genetic polymorphism sex investigation of race, Kazak, the Dai nationality, Miao ethnic group and five, the Yao nationality, the personal discrimination of accumulation is respectively
0.999999999957th, 0.999999999990,0.999999999974,0.999999999875 and 0.999999999966,
There is higher cumulative individual discrimination.
2nd, the method for the present invention and system are applied to extensive Chinese population, can degrade for forensic science test in laboratory
Sample provides effective technical support, while reducing testing cost, becoming replacement STR detection architecture carries out DNA sample analysis
Another powerful measure.
3rd, the scheme that the present invention is provided effectively can realize the deduction to unknown sample source from gene level, be Chinese population
Individual identification and the identification of parental right relation etc. provide accurate scientific basis.
Description of the drawings
Fig. 1 is using the parting collection of illustrative plates of composite amplification system test stone DNA9947 sample.
Specific embodiment
The fresh peripheral venous samples of 421 parts of independent individuals used in following examples, wherein 94 parts of Han nationality in Beijing, cloud
97 parts of southern the Dai nationality, 95 parts of Kazak ethnic population, 69 parts of Guangxi Miao, 66 parts of the Guangxi Yao nationality, carried by Material Evidence Identification Center, Ministry of Public Security
For.
DNTP (10mM), 10 × PCR buffer (15mM Mg used in following examples2+)、10×PCR buffer
(15mM Mg2+), quick start in enzyme Hotstar Taq plus (5U/ μ l), human standard product (9947,1ng/ μ l), molecular weight
Mark Typer500 is purchased from Material Evidence Identification Center, Ministry of Public Security, and POP7 running gel, deionized formamide are purchased from American AB company,
Fluorescence labeling PCR primer is synthesized by Shanghai life work.9700 type PCR instrument, 3130XL genetic analyzer are American AB company.
Embodiment 1, the checking to the method and system accuracy for carrying out individual identification to unknown sample of the present invention
In the present embodiment, the unknown sample is the fresh peripheral venous samples of 421 parts of independent individuals, wherein Bei Jinghan
94 parts of race, 97 parts of Yunnan the Dai nationality, 95 parts of Kazak ethnic population, 69 parts of Guangxi Miao, 66 parts of the Guangxi Yao nationality, it is known that its individuality comes
Source, but set in the implementation process of the embodiment of the present application 1 its individuality source unknown, which is entered using the application method and system
Row individual identification, including：
1) extract, using the DNA in the system of the present invention, the DNA that system extracts unknown sample, 2) using the system of the present invention
In compound detection system obtain the DNA and include that 30 InDel locus and 1 sex identification locus Amelogenin exist
The interior genotyping result of totally 31 locus, 3) system is inferred described in the system using the present invention, according to 31 bases of unknown sample
Because the genotype of seat carries out individual identification.
In the present embodiment, the compound detection system includes unknown sample DNA, 31 locus, and amplimer, institute
Compound detection system is stated for the DNA being obtained including totally 31 including 30 InDel locus and 1 sex identification locus
The genotyping result of locus, 30 InDel locus be rs10629077, rs2308026, rs361519,
rs2307652、rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、
rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、
rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、
Rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and rs140864；The amplimer by with institute
The one-to-one 31 pairs of amplimers composition of 31 locus is stated, the amplimer is SEQ ID No.1 to SEQ in sequence table
The nucleotide sequence of ID No.62.
1st, the DNA of to be detected sample is extracted as template
According toDNA Blood Midi Kit specification (Qiagen, Germany) extracts blood sample DNA.All
After DNA is all through Nanodrop2000c (Thermo Scientific, the U.S.) quantitation, 0.5ng/ μ l- is diluted to ultra-pure water
1ng/ μ l is used.
2nd, the parting of 31 locus is carried out using the compound detection system, including：The unknown sample DNA for extracting is made
For template；Multiplexed PCR amplification reaction is carried out to the DNA profiling for extracting using the amplimer, to obtain amplified production；To expand
Increase the genotyping result that product utilization genetic analyzer determines 31 locus.
Detailed process is as follows：
2.1st, primer pond configuration
The configuration in amplimer pond, is the one-to-one 31 pairs of amplifications of 31 locus in wherein described amplimer
Primer, in the present embodiment, it is preferred that the amplimer of 31 locus is SEQ ID No.1 to SEQ ID in sequence table
The nucleotide sequence of No.62；The various primer sequences that the present invention is provided are by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Synthesis.
Synthetic primer is diluted to 100 μM with 1 × TE buffer solution, by the upstream and downstream primer of 31 locus according to
Lower volume and concentration are mixed as 31 weights PCR primer pond (i.e. PrimerMix).
2.2nd, multi-PRC reaction
The present embodiment carries out multi-PRC reaction using 9700 type PCR amplification instrument.
(1) configuration PCR mix (10 μ L system), as shown in table 5 below.
Table 5
(2) amplification program
The thermal circulation parameters of PCR amplification procedure are：95℃11min；94 DEG C of 30s, 60 DEG C of 120s, 72 DEG C of 90s, totally 30 are followed
Ring；60 DEG C of extension 60min.
2.3rd, PCR primer parting
Take 1 μ L PCR primer and 9.5 μ L deionized formamides, Typer500 internal standard to mix, ice bath immediately after 95 DEG C of 3min
5min.Amplified production is passed through using ABI 3130XL type genetic analyzerID v3.2 software is carried out point
Analysis, obtains the genotype of 31 locus.
2.4th, interpretation of result
In order to the accuracy of genotyping result is verified, 50 parts of DNA sample are randomly selected from 421 parts of DNA sample, to 31 bases
Because seat is sequenced (sequencing of Beijing Mai Aodeen bio tech ltd), obtained using the compound detection system of the present embodiment
All of genotyping result all consistent with sequencing result, uniformity reaches 100%, and this result is demonstrate,proved compound detection system of the present invention and divided
Type result is accurate.
While the use of standard DNA 9947 is that template enters performing PCR amplification using the system of the present invention, and to its 31 locus
Parting is carried out, genotyping result is as shown in Figure 1, consistent with DNA9947 sequencing result.
3rd, individual identification is carried out according to the genotype of 31 locus of unknown sample
The individual source results of the above-mentioned 421 parts of samples obtained by the present embodiment method, with individual source knot known to which
Fruit is consistent, illustrates that the inventive method can carry out individual identification to unknown sample.
In the detecting system of the present invention of embodiment 2,30 InDel locus are in the balance check of different nationalities
The genotyping result of the independent individuals of 421 people, and base is obtained using present system according to 1 methods described of embodiment
Individual identification rate (DP value, be shown in Table 6) in 30 locus in various nationalities, calculates 30 autosome InDel locus and exists
The national cumulative individual discrimination (TDP) of Han nationality, Kazak, the Dai nationality, Miao ethnic group and five, the Yao nationality.
Table 6
Cumulative individual discrimination computing formula is：
TDP=1- (1-DP1)(1-DP2)(1-DP3)…(1-DPK), wherein DPKDP value for k-th locus.
The detecting system of the application is substantially increased to individual cumulative individual discrimination, in Han nationality, Kazak, the Dai Nationality
In the national genetic polymorphism sex investigation of race, Miao ethnic group and five, the Yao nationality, the personal discrimination of accumulation respectively 0.999999999957,
0.999999999990th, 0.999999999974,0.999999999875 and 0.999999999966, many with higher heredity
State property and cumulative individual discrimination.
Sequence table
<110>Material Evidence Identification Center, Ministry of Public Security
<120>A kind of method and system for carrying out individual identification to unknown sample
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<400> 50
ccagcaaaca gaacacaagg 20
<210> 51
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 51
gtatggcctc ctccacagag 20
<210> 52
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 52
acagcccacc agagcactac 20
<210> 53
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 53
accgggtgtg cattctacat 20
<210> 54
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 54
gtgcctggcc aagaaaatta 20
<210> 55
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 55
cagatcagca aaggctggta 20
<210> 56
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 56
gtttggaaag aaaggcagga 20
<210> 57
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 57
gcacccagcc ttctccttat 20
<210> 58
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 58
gtggttcatt tcagactaca actca 25
<210> 59
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 59
accacaggca aatcctgaag 20
<210> 60
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 60
gggttaggga ggttggttga 20
<210> 61
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 61
ccctgggctc tgtaaagaa 19
<210> 62
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 62
gagcttaaac tgggaagctg 20
Claims (8)
1. a kind of method that individual identification is carried out to unknown sample, it is characterised in that the method includes：
1) DNA of unknown sample is extracted；
2) DNA is obtained including totally 31 bases including 30 InDel locus and 1 sex identification locus Amelogenin
Because of the genotyping result of seat, 30 InDel locus are rs10629077, rs2308026, rs361519, rs2307652,
rs16671、rs33948716、rs8190507、rs1610937、rs2307689、rs2307976、rs1305056、
rs2067353、rs1610963、rs2307632、rs17878444、rs140847、rs2307554、rs2308020、
rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、rs2307553、
Rs16438, rs2307981, rs4646006, rs2308072 and rs140864；
3) individual identification is carried out according to the genotype of 31 locus of unknown sample.
2. method according to claim 1, it is characterised in that 2) include using with one a pair of 31 locus
The step of 31 pairs of amplimers that answers are expanded to which to obtain amplified production；The amplimer is SEQ ID in sequence table
The nucleotide sequence of No.1 to SEQ ID No.62.
3. method according to claim 2, it is characterised in that after wherein 2) being additionally included in acquisition amplified production, using something lost
Pass analyzer analyze the amplified production, with obtain 31 locus genotype the step of.
4. a kind of system that individual identification is carried out to unknown sample, it is characterised in that the system includes that DNA extracts system, multiple
Detection architecture is closed, and infers system；
The DNA extracts system is used for extracting the DNA of unknown sample；
The compound detection system includes 30 InDel locus and 1 sex identification locus for obtaining the DNA
Amelogenin in the genotyping result of interior totally 31 locus, 30 InDel locus are rs10629077,
rs2308026、rs361519、rs2307652、rs16671、rs33948716、rs8190507、rs1610937、
rs2307689、rs2307976、rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、
rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、
Rs3047269, rs2067294, rs2307553, rs16438, rs2307981, rs4646006, rs2308072 and
rs140864；
The deduction system is used for carrying out individual identification according to the genotype of 31 locus of unknown sample.
5. system according to claim 4, it is characterised in that the compound detection system be used for using and 31 bases
Because the one-to-one 31 pairs of amplimers of seat are expanded to which to obtain amplified production, the amplimer is in sequence table
The nucleotide sequence of SEQ ID No.1 to SEQ ID No.62.
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 31 locus.
7. a kind of compound detection system, it is characterised in that the system includes unknown sample DNA, 31 locus, and amplification
Primer,
The compound detection system includes 30 InDel locus and 1 sex identification locus for obtaining the DNA
Amelogenin in the genotyping result of interior totally 31 locus, 30 InDel locus are rs10629077,
rs2308026、rs361519、rs2307652、rs16671、rs33948716、rs8190507、rs1610937、
rs2307689、rs2307976、rs1305056、rs2067353、rs1610963、rs2307632、rs17878444、
rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、
rs3047269、rs2067294、rs2307553、rs16438、rs2307981、rs4646006、rs2308072、rs140864；
By constituting with the one-to-one 31 pairs of amplimers of 31 locus, the amplimer is sequence to the amplimer
The nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in list.
8. a kind of detection kit, it is characterised in that including the compound detection system described in claim 7.
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| CN107400713A (en) * | 2017-08-18 | 2017-11-28 | 公安部物证鉴定中心 | A kind of method and system that the Qinghai-Tibet Tibetan Population individual of China is identified in 27 colonies |
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| CN108531572A (en) * | 2018-03-08 | 2018-09-14 | 北京爱普益医学检验中心有限公司 | It is a kind of it is antenatal detection progeny genotypes method and application |
| CN112011622A (en) * | 2019-05-29 | 2020-12-01 | 公安部物证鉴定中心 | Method and system for analyzing non-east Asia and European population sources of individuals with unknown sources |
| CN113322329A (en) * | 2021-05-14 | 2021-08-31 | 公安部物证鉴定中心 | DIP rapid amplification detection reagent for fully integrated microfluidic chip and application thereof |
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