CN106480198B - A kind of method and system carrying out individual identification to unknown sample - Google Patents

Abstract

The present invention provides a method and system for individual identification of unknown specimens. The method includes extracting DNA from unknown specimens, and obtaining a total of 31 genes including 30 InDel loci and 1 sex identification locus Amelogenin from the DNA.座的分型结果，所述30个InDel基因座为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; individual identification was performed based on the genotypes of 31 loci of unknown samples. The scheme of the present invention can utilize the InDel loci to realize individual identification of unknown specimens in the Chinese population.

Description

A method and system for individual identification of unknown samples

technical field

The present invention relates to a method and system for individual identification, in particular to a method and system for individual identification of unknown specimens.

Background technique

Insertion-deletion polymorphism (insertion-deletion, InDel) is a special type of two-allelic polymorphism formed by the insertion or deletion of a DNA segment. Compared with STR and SNP, InDel has the following characteristics: (1) widely distributed in the entire genome; (2) due to single mutation events, and the frequency of occurrence is low, relatively stable after occurrence, and not easy to recur; (3) It can become the ancestral information site and determine the geographical source; (4) InDel can be amplified in smaller amplicons, which is suitable for the detection of degraded DNA; (5) InDel can be detected by using the existing equipment of the forensic DNA laboratory Genotyping; (6) InDel can be applied to automation and high-throughput technologies at the same time.

Therefore, InDel has attracted the attention of scholars at home and abroad, and many scholars have established a composite system suitable for the study of polymorphisms in their own nation. However, due to the diversity of alleles of various ethnic groups in the Chinese population, how to establish a complex amplification system based on the InDel locus for forensic DNA identification of a wide range of Chinese populations, making it an alternative to the STR detection system for analyzing DNA samples Another powerful means to become a problem to be solved.

Contents of the invention

The present invention provides a method for individual identification of unknown specimens, which can obtain the typing results of 31 loci including 30 InDel loci and 1 sex identification locus in unknown specimens, thereby realizing the identification of unknown Individual identification of specimens.

The present invention also provides a system for individual identification of unknown samples, through which the accurate typing of unknown samples for the above 31 loci can be realized, so as to perform individual identification of unknown samples.

The present invention also provides a composite detection system, which can accurately obtain the typing results of 31 loci in unknown samples including 30 InDel loci and 1 sex identification locus.

The present invention also provides a detection kit, including the composite detection system.

A method for individual identification of unknown samples provided by the present invention, the method comprising:

1) Extract the DNA of unknown samples;

2) Obtain the typing results of 31 loci in the DNA including 30 InDel loci and 1 sex identification locus Amelogenin, the 30 InDel loci 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和rs140864；

3) Individual identification was performed based on the genotypes of 31 loci of unknown specimens.

In the solution of the present invention, the 31 loci are the applicants’ comprehensive analysis of the living environment and ethnic origin of the Chinese population, and the investigation of the differences in phenotypic characteristics of ethnic populations in various regions, including appearance characteristics, physiological indicators, etc., For these differences, literature and network database investigations are carried out, and the combination of specific loci that can be used for individual identification and paternity identification is obtained on the basis of existing research. The unknown samples can be samples such as blood samples, exfoliated cells, bones, teeth, semen plaques and oral swabs from the human body, and the individual sources of these samples are unknown.

In a specific embodiment of the present invention, said 2) includes the step of using 31 pairs of amplification primers corresponding to said 31 loci to amplify it to obtain amplification products; said amplification primers It is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in the sequence listing.

In another specific embodiment of the present invention, 2) further includes the step of analyzing the amplified product with a genetic analyzer to obtain the genotypes of the 31 loci after obtaining the amplified product. In the solution of the present invention, the genetic analyzer can be a genetic analyzer routinely used by those skilled in the art, such as ABI3130 or ABI3500 genetic analyzer, through ID-X software or other GeneMapper software etc. analyze the genotypes of the 31 loci in the PCR amplification product.

A system for individual identification of unknown samples provided by the present invention, the system includes a DNA extraction system, a composite detection system, and an inference system; the DNA extraction system is used to extract DNA from unknown samples; the composite detection The system is used to obtain the typing results of 31 loci in the DNA including 30 InDel loci and 1 sex identification locus Amelogenin. The 30 InDel loci 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和rs140864 ;

The inference system is used for individual identification based on the genotypes of 31 loci in unknown specimens.

In a specific embodiment of the present invention, the composite detection system is used to amplify the 31 loci with 31 pairs of amplification primers corresponding to one-to-one to obtain amplification products, and the amplification primers It is the nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in the sequence listing.

Furthermore, the composite detection system is also used to analyze the amplification products with a genetic analyzer after obtaining the amplification products, so as to obtain the genotypes of the 31 loci.

A compound detection system provided by the present invention, the system includes unknown sample DNA, 31 loci, and amplification primers, and the compound detection system is used to obtain the DNA including 30 InDel loci and 1 sex The typing results of a total of 31 loci including Amelogenin, the 30 InDel loci are rs10629077, rs2308026, rs361519, rs2307652, rs16671, rs33948716, rs8190507, rs1610937, rs2307689, rs2361, rs2307976, rs1302rs906 rs2307632、rs17878444、rs140847、rs2307554、rs2308020、rs1610902、rs2307839、rs2307708、rs2308115、rs3047269、rs2067294、rs2307553、rs16438、rs2307981、rs4646006、rs2308072和rs140864；所述扩增引物由与所述31个基因座一一对应Composed of 31 pairs of amplification primers, the amplification primers are the nucleotide sequences of SEQ ID No.1 to SEQ ID No.62 in the sequence listing.

In the solution of the present invention, the DNA polymerase may be one or more of Fast Start DNA polymerase, Taq DNA polymerase, and Hotstart DNA polymerase.

The present invention also provides a detection kit, including the composite detection system.

In the scheme of the present invention, the method for typing the 31 loci using the composite detection system includes: 1) using the extracted DNA of unknown samples as a template; 2) using the amplification primer pair as a template; The DNA of the unknown sample of the template is subjected to multiple PCR amplification reactions to obtain amplification products; 3) the amplification products are analyzed by a genetic analyzer to obtain the typing results of 31 loci.

The inventive solution has the following advantages:

1, the method and system of the present invention adopt specific 31 loci to carry out individual identification, and these loci are in the genetic polymorphism investigation of five nationalities of Han nationality, Kazak nationality, Dai nationality, Miao nationality and Yao nationality, and cumulative individual identification rate is respectively 0.999999999957, 0.999999999990, 0.999999999974, 0.999999999875 and 0.999999999966 have a higher cumulative individual recognition rate.

2. The method and system of the present invention are applicable to a wide range of Chinese people, can provide effective technical support for forensic science laboratories to detect degraded samples, and reduce detection costs at the same time, becoming another powerful means to replace the STR detection system for DNA sample analysis .

3. The solution provided by the present invention can effectively realize the inference of the source of unknown samples from the genetic level, and provide accurate scientific basis for individual identification and parental relationship identification of the Chinese population.

Description of drawings

Fig. 1 is the typing map of the standard DNA9947 sample tested by the multiplex amplification system.

Detailed ways

The fresh peripheral venous blood samples of 421 unrelated individuals used in the following examples, including 94 samples of Han nationality in Beijing, 97 samples of Dai nationality in Yunnan, 95 samples of Kazak nationality in Xinjiang, 69 samples of Miao nationality in Guangxi, and 66 samples of Yao nationality in Guangxi, were provided by the Physical Evidence Identification Center of the Ministry of Public Security .

dNTP (10mM), 10×PCR buffer (15mM Mg ²⁺ ), 10×PCR buffer (15mM Mg ²⁺ ), quick start enzyme Hotstar Taq plus (5U/μl), human standard (9947 , 1ng/μl), molecular weight internal standard Typer500 were purchased from the Physical Evidence Identification Center of the Ministry of Public Security, POP7 electrophoresis gel and deionized formamide were purchased from AB Company in the United States, and fluorescently labeled PCR primers were synthesized by Shanghai Sangong. Model 9700 PCR instrument and 3130XL genetic analyzer are from AB Company of the United States.

Embodiment 1. Verification of the accuracy of the method and system for individual identification of unknown samples of the present invention

In this example, the unknown samples were 421 fresh peripheral venous blood samples from unrelated individuals, including 94 samples from the Han nationality in Beijing, 97 samples from the Dai nationality in Yunnan, 95 samples from the Kazak nationality in Xinjiang, 69 samples from the Miao nationality in Guangxi, and 66 samples from the Yao nationality in Guangxi. Its individual source is known, but its individual source is set to be unknown during the implementation of Example 1 of this application, and its individual identification is carried out by using the method and system of this application, including:

1) Utilize the DNA extraction system in the system of the present invention to extract DNA from unknown specimens, 2) use the composite detection system in the system of the present invention to obtain the DNA including 30 InDel loci and 1 sex identification locus Amelogenin 3) use the inference system described in the system of the present invention to perform individual identification based on the genotypes of the 31 loci in unknown samples.

In this embodiment, the composite detection system includes DNA from unknown samples, 31 loci, and amplification primers, and the composite detection system is used to obtain the DNA including 30 InDel loci and 1 sex identification locus在内共31个基因座的分型结果，所述30个InDel基因座为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和rs140864；所述扩增引物由与所述31个基因座一一对应的31对扩The amplification primer is composed of the nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in the sequence listing.

1. Extract the DNA of the sample to be tested as a template

according to DNA Blood Midi Kit manual (Qiagen, Germany) was used to extract DNA from blood samples. All DNA was quantified by Nanodrop2000c (Thermo Scientific, USA), diluted with ultrapure water to 0.5ng/μl-1ng/μl for use.

2. Using the composite detection system to carry out the typing of 31 loci, including: using the extracted unknown sample DNA as a template; using the amplification primers to perform multiple PCR amplification reactions on the extracted DNA template to obtain amplified Amplified products; the amplified products were determined using a genetic analyzer to determine the typing results of 31 loci.

The specific process is as follows:

2.1. Primer pool configuration

The configuration of the amplification primer pool, wherein the amplification primers are 31 pairs of amplification primers corresponding to the 31 loci one to one, in this embodiment, preferably, the amplification primers of the 31 loci are The nucleotide sequences of SEQ ID No.1 to SEQ ID No.62 in the sequence listing; various primer sequences provided by the present invention were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

The synthesized primers were diluted to 100 μM with 1×TE buffer, and the upstream and downstream primers of 31 loci were mixed according to the following volumes and concentrations as a 31-fold PCR primer pool (ie, PrimerMix).

2.2. Multiplex PCR reaction

In this example, a 9700-type PCR amplification instrument was used to perform multiple PCR reactions.

(1) Configure the PCR mix (10 μL system), as shown in Table 5 below.

table 5

(2) Amplification program

The thermal cycle parameters of the PCR amplification process were: 95°C for 11 minutes; 94°C for 30s, 60°C for 120s, and 72°C for 90s, a total of 30 cycles; 60°C for 60 minutes.

2.3. PCR product typing

Mix 1 μL of PCR product with 9.5 μL of deionized formamide and Typer500 internal standard, and immediately put it in ice bath for 5 minutes at 95°C for 3 minutes. ABI 3130XL Genetic Analyzer was used to pass the amplification product ID v3.2 software was used for analysis to obtain the genotypes of the 31 loci.

2.4. Result analysis

In order to verify the accuracy of the typing results, 50 DNA samples were randomly selected from 421 DNA samples, and 31 loci were sequenced (sequenced by Beijing Maiodeen Biotechnology Co., Ltd.), using the composite detection system of this example All the typing results obtained are consistent with the sequencing results, and the consistency reaches 100%, which proves that the typing results of the composite detection system of the present invention are accurate.

At the same time, the standard DNA9947 was used as a template to carry out PCR amplification with the system of the present invention, and its 31 loci were typed. The typing results are shown in Figure 1, which is consistent with the DNA9947 sequencing results.

3. Individual identification based on the genotypes of 31 loci of unknown samples

The individual origin results of the above 421 specimens obtained by the method of this embodiment are consistent with the known individual origin results, indicating that the method of the present invention can perform individual identification on unknown specimens.

Example 2 The balance test of 30 InDel loci in different nationalities in the detection system of the present invention

The system of the present invention is used to obtain the typing results of unrelated individuals of 421 cases according to the method described in Example 1, and based on the individual recognition rate (DP value, see Table 6) of 30 loci in each ethnic group, calculate 30 common Cumulative individual recognition rate (TDP) of chromosome InDel loci in Han, Kazak, Dai, Miao and Yao nationalities.

Table 6

The formula for calculating the cumulative individual recognition rate is:

TDP=1-(1-DP ₁ )(1-DP ₂ )(1-DP ₃ )...(1-DP _K ), where DP _K is the DP value of the kth locus.

The detection system of the present application has greatly improved the cumulative individual identification rate of individuals. In the genetic polymorphism investigation of the five nationalities of Han, Kazak, Dai, Miao and Yao, the cumulative individual identification rates were 0.999999999957, 0.999999999990, 0.999999999974, 0.999999999974, 0.999999999875 and 0.999999999966, with high genetic polymorphism and cumulative individual recognition rate.

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Claims (8)

1. the method that the unknown sample of a kind of pair of Chinese population carries out individual identification, which is characterized in that this method comprises:

1) DNA of the unknown sample is extracted；

2) DNA totally 31 bases including 30 InDel locus and 1 sex identification locus Amelogenin are obtained Because of the genotyping result of seat, 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 described 31 locus of unknown sample.

2. the method according to claim 1, wherein 2) described includes using and described 31 locus, one a pair The step of 31 pairs of amplimers answered expand to obtain amplified production it；The amplimer is SEQ ID in sequence table The nucleotide sequence of No.1 to SEQ ID No.62.

3. according to the method described in claim 2, it is characterized in that, wherein 2) further including using something lost after obtaining amplified production It passes analyzer and analyzes the amplified production, the step of genotype to obtain 31 locus.

4. a kind of system for carrying out individual identification to unknown sample, which is characterized in that the system comprises DNA extraction systems, multiple Detection architecture is closed, and infers system；

The DNA extraction system is used to extract the DNA of unknown sample；

The compound detection system includes 30 InDel locus and 1 sex identification locus for obtaining the DNA The genotyping result of Amelogenin totally 31 locus inside, 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 deduction system is used to carry out individual identification according to the genotype of 31 locus of unknown sample.

5. system according to claim 4, which is characterized in that the compound detection system is used to use and 31 bases Because the one-to-one 31 pairs of amplimers of seat expand it 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, which is characterized in that the compound detection system is also used 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, which is characterized in that the system includes unknown sample DNA and amplimer,

The compound detection system includes 30 InDel locus and 1 sex identification locus for obtaining the DNA The genotyping result of Amelogenin totally 31 locus inside, 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,rs140864；

For the amplimer by forming with the one-to-one 31 pairs of amplimers of 31 locus, the amplimer is sequence The nucleotide sequence of SEQ ID No.1 to SEQ ID No.62 in list.

8. a kind of detection kit, which is characterized in that including compound detection system as claimed in claim 7.