CN112143819A - Genetic marker combination and special primer combination for individual identity recognition - Google Patents
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Abstract
The invention discloses a genetic marker combination and a special primer combination for individual identity identification. The invention firstly protects a genetic marker combination which consists of 24 SNPs, and the individual identification of people can be realized by detecting the group of genetic markers. The specific primer group A consists of 48 primers; the 48 primers are sequentially shown as a sequence 1 to a sequence 48 in a sequence table. The specific primer group B consists of 48 primers; the 48 primers are obtained by adding sample labels at the 5' ends of the 48 primers of the specific primer group A. The invention can be used for individual identification of people, enriches effective information for identity confirmation of people, can be used for genetic identification of people, sample source identification and the like, and has important application and popularization values.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a genetic marker combination and a special primer combination for individual identity identification.
Background
Each cell has 23 pairs (46) of chromosomes, containing about 30 hundred million nucleotides, and each sperm and egg is unique and unique because it undergoes homologous chromosome exchange and non-homologous chromosome recombination prior to formation, and it can be said that no one has exactly the same gene sequence as another, except for the homozygote twins, which is a genetic polymorphism in humans. The identification of individuals by using human genetic polymorphisms is the most common technical means in modern medicine.
At present, the most important means for the biological identification of human is to adopt STR (short distance repeat) technology. STR sequences are polymorphic in a population, i.e., different people may have different numbers of repeats of the same STR sequence, i.e., different people may have different STRs for the same STR. DNA detection is carried out by using 13 core STR loci of international standard, and by integrating the locus information, the individual recognition rate of an individual exceeds one billion, namely STR types of two individuals are not repeated in 1000 billion people, and identity identification of one person can be completely carried out. However, as a second generation genetic marker, STRs also have a short panel in terms of individual identification, and after PCR amplification, STRs are subjected to electrophoresis to determine the length of STRs, which has limitations such as high requirements for samples and detection instruments, complex technology, long time consumption, difficult result interpretation, low throughput, and difficulty in automation.
Single Nucleotide Polymorphism (SNP) mainly refers to a DNA sequence polymorphism caused by variation of a single nucleotide at the genome level. It is the most common one of the human heritable variations, accounting for over 90% of all known polymorphisms. At present, the SNP technology has wide application in the aspects of animal and plant variety identification, gene positioning, linkage analysis and the like. Identification of individuals can also be achieved entirely using appropriate combinations of SNPs. Compared with the second generation genetic marker STR technology, the SNP technology as the third generation genetic marker has unique advantages. First, in the human genome, the number of SNPs is large, 1 in each 300 base pair on average, and the total number is estimated to be 300 ten thousand or more. Secondly, SNP is a two-state marker, and there are at least three combinations of genotypes as a SNP site. In addition, SNPs also have high genetic stability compared to genetic markers of repetitive sequences such as STRs. Most importantly, the detection of SNPs can be directly confirmed by sequencing and is extremely amenable to computer automation. The detection of SNP has low requirements on samples, and because SNP only involves the change of a single base, the detection can be carried out by amplifying the length of 100bp generally, so for some DNA samples which are seriously degraded, the identification can be difficult to realize by STR technology, and the identification can be easily realized by SNP detection. Therefore, in the aspect of individual identification, a new individual identification technology based on SNP is needed to meet the identification requirements of different samples.
Disclosure of Invention
The invention aims to provide a genetic marker combination and a special primer combination for individual identification, in particular to a group of genetic markers (SNP markers) screened and found by a discoverer, the individual identification of people can be realized by detecting the group of genetic markers, and further, the invention develops a primer combination and a kit for detecting the group of genetic markers.
The invention firstly protects a genetic marker combination which consists of 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
The invention also protects a gene identity card which records the genotype information of 24 SNPs of a human to be tested; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
The invention also protects a two-dimensional code which can read the genotype information of 24 SNPs of a human subject; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
The invention also protects the application of the genetic marker combination, the gene identity card or the two-dimensional code in the identification of individual human identities.
The invention also provides the application of the substance for detecting 24 SNPs in the preparation of the kit; the use of the kit is for identifying human individuals; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
The invention also provides the application of the substance for detecting 24 SNPs in human individual identification; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
The substance for detecting 24 kinds of SNPs is a substance for detecting genotypes of 24 kinds of SNPs.
The substance for detecting 24 SNPs can be specifically a specific primer group A and/or a specific primer group B.
The invention also protects a specific primer group A, which consists of 48 primers; the 48 primers are sequentially shown as a sequence 1 to a sequence 48 in a sequence table. In the specific primer group A, 48 primers are mixed and packaged. In the specific primer group A, 48 primers are in equal molar ratio.
The invention also protects a specific primer group B, which consists of 48 primers; the 48 primers are obtained by adding sample labels at the 5' ends of the 48 primers of the specific primer group A. For a certain sample to be detected, the upstream primers in the specific primer group B have the same sample label, and the downstream primers in the specific primer group B have the same sample label. Thereby making the sample tag of the amplification product unique for each sample. Illustratively, the sample labels are shown in table 6 in example 3. And in the specific primer group B, 48 primers are mixed and packaged. In the specific primer group B, 48 primers are in equal molar ratio.
The specific primer group A and the specific primer group B are collectively referred to as a specific primer group.
The invention also protects the application of the specific primer group A and/or the specific primer group B, which is (a), (B) or (c) as follows:
(a) the application of the gene in identifying the genotypes of 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509;
(b) the application in the preparation of the kit; the use of the kit is for human individual identification.
(c) The application in human individual identification.
The invention also provides a method for identifying the individual, which comprises the following steps:
(1) detecting the genotype of a human to be tested based on 24 SNPs to obtain the corresponding 24 genotype information of the 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509;
(2) the human subject was identified by the genotype information of the 24 SNPs.
The method further comprises the steps of: and making the 24 genotype information into a gene identity card, and identifying the testee through the gene identity card.
The invention also provides a method for identifying the individual, which comprises the following steps:
(1) detecting the genotype of a human to be tested based on 24 SNPs to obtain the corresponding 24 genotype information of the 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509;
(2) and making the genotype information of the 24 SNPs into a gene identity card, and identifying the testee through the gene identity card.
SNP typing methods are various, such as Sanger sequencing, fluorescent quantitative PCR, competitive allele-specific PCR (KASP), mass spectrometry, high throughput sequencing, etc., and only one specific method, i.e., sequencing, is described in the present invention by way of example. In fact, one skilled in the art can use any of the methods in the prior art, and the present invention is not limited thereto.
The method for detecting the genotype of the human subject based on the 24 SNPs comprises the following steps: and (3) taking the genome DNA of a human to be tested as a template, carrying out PCR amplification by adopting the specific primer group A, and obtaining 24 corresponding genotype information of 24 SNPs through sequencing.
The method for detecting the genotype of the human subject based on the 24 SNPs comprises the following steps: and (3) taking the genome DNA of a human to be tested as a template, carrying out first round PCR amplification by adopting the specific primer group A, carrying out second round PCR amplification by adopting the specific primer group B, and obtaining 24 corresponding genotype information of 24 SNPs through high-throughput sequencing.
The reaction system for the first round of PCR amplification procedure is specifically shown in Table 3 in the examples.
The reaction sequence of the first round PCR amplification procedure is specifically shown in Table 4 in the examples.
The reaction system for the second round of PCR amplification procedure is specifically shown in Table 5 in the examples.
The reaction sequence of the second round of PCR amplification procedure is specifically shown in Table 7 in the examples.
The sequencing platform for high-throughput sequencing includes, but is not limited to, a sequencing platform of Illumina company, such as Nextseq 500, Hiseq X10 or Novaseq 6000.
Any of the above people may be Han nationality.
Any of the above people may be Chinese Han nationality.
The rs number is the reference SNP ID, and for SNP loci adopting other numbering modes, if the SNP loci and the SNP in the invention indicate the same gene locus, the rs number is within the protection scope of the invention.
The invention can be used for individual identification of people, enriches effective information for identity confirmation of people, can be used for genetic identification of people, sample source identification and the like, and has important application and popularization values.
Drawings
Fig. 1 is an exemplary two-dimensional code.
FIG. 2 is an exemplary identity alignment.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 screening of SNP marker
The basic principle of the inventor for screening SNP: the selected SNP must be located in the non-coding region or non-gene region of the gene, the selected SNPs are all on an autosome, in principle, only 1 SNP site is selected on one autosome, 2 SNP sites are selected on individual autosome (the distance between two sites should be up to 10M to ensure independent representativeness of each SNP), and the frequency of the selected SNP's secondary allele (MAF) in the CHB and CHS population in the thousand people genome project database must be greater than 0.3.
Through a large amount of screening and verification, 24 SNP markers are finally obtained. None of the selected SNPs have clear functional information in the existing literature, and the arrangement of genotype information of the selected SNPs is theoretically unique among 2000 billion of people. The combination of SNP site genotypes in the invention is only used for identification of individuals, and the selected SNP sites do not contain any information related to the characteristics (including sex), health and diseases of the individuals so as to avoid revealing individual privacy.
The information of the 24 SNP markers is shown in Table 1.
TABLE 1
rs number is reference SNP ID.
Example 2 development of multiplex primer set for detecting the 24 SNP markers
And obtaining the multiple primer groups through a large amount of screening and combination effect verification. The multiplex primer set consists of 24 primer pairs (each primer pair includes an upstream primer F and a downstream primer R, and thus 24 primer pairs, i.e., 48 primers), each primer pair being used for amplifying 1 SNP marker and its peripheral region. The multiplex primer set can simultaneously amplify the 24 SNP markers and the peripheral regions thereof in the table 1, thereby realizing the detection.
The names and nucleotide sequences of the individual primers in the multiplex primer set are shown in Table 2.
TABLE 2
Example 3 establishment of method (Gene sequencing typing method based on multiplex PCR library construction)
Blood sample: venous blood of the test subjects.
Firstly, extracting DNA of a sample
DNA of the Blood sample was extracted using QIAamp DNA Blood Mini Kit (QIAGEN, cat No. 69504) DNA extraction Kit. The method comprises the following specific steps: adding 200 μ l of blood sample into 1.5ml centrifuge tube, adding 200 μ l of lysate (BUFFER AL; provided by kit) and 20 μ l of proteinase K (10 mg/ml; provided by kit), digesting at 70 deg.C for 15min, and centrifuging; then adding 200 mul of absolute ethyl alcohol, reversing, uniformly mixing and centrifuging; then transferring the mixture to a centrifugal column, centrifuging the mixture at 12000rpm for 1min, and removing effluent liquid; then adding 500. mu.l of washing solution I (AW 1; provided by kit), centrifuging at 12000rpm for 1min, and discarding the effluent; then adding 500 mul of washing liquid I, centrifuging at 12000rpm for 1min, and discarding the effluent liquid; then adding 500. mu.l of washing solution II (AW 2; provided by kit), centrifuging at 12000rpm for 1min, and discarding the effluent; centrifuging the centrifugal column at 12000rpm for 3min, and spin-drying; then transferring the centrifugal column into a new centrifugal tube, and airing for 5min to remove residual ethanol; add 65. mu.l ddH to the air dried spin column2And O, centrifuging at 12000rpm for 2min, and collecting a centrifugate part to obtain the DNA solution.
DNA quantification was performed using a Qubit nucleic acid quantification apparatus and the DNA concentration was recorded. The concentration of DNA should be 20 ng/. mu.l or more, and if it is lower than this concentration, concentration treatment may be performed.
Two, multiple capture and library building
1. Performing a first round of PCR amplification
The reaction system was prepared and then subjected to a first round of PCR amplification procedure.
The resulting DNA solution from each blood sample was subjected to two replicates (i.e., 2 reaction systems were prepared). Each reaction system is shown in Table 3.
TABLE 3
| Components | Volume of |
| Sample DNA | 1μl |
| PrimerMix | 2.7μl |
| 2×KAPA_Enzyme Mix | 5μl |
| ddH2O | 1.3μl |
| Total volume | 10μl |
PrimerMix: 96 ul of multiplex primer stock and 404 ul of ddH2O mixed to give 500. mu.l PrimerMix. In PrimerMix, the primer concentration was always 3.84. mu.M (i.e., each of the 48 primersThe concentrations of all substances were 0.08. mu.M).
Mother solution of multiple primers: mu.l of each of 48 primers (each primer concentration was 20. mu.M) was mixed to obtain 96. mu.l of a multiplex primer stock solution.
The reaction procedure is shown in Table 4.
TABLE 4
2. Purification of PCR products
(1) After completion of step 1, the amplification products from both reaction systems were combined and 20. mu.lddH was added2O (Total volume at this time 40. mu.l)
(2) After the step (1) is finished, adding 0.6 times volume of AMPure XP Beads (namely adding 24 mu l), blowing and beating up and down by using a pipette gun to fully and uniformly mix the amplified product and the magnetic Beads, and standing for 2min at room temperature.
(3) After step (2) was completed, the magnetic beads were adsorbed by a magnetic rack until the solution was clarified, and the supernatant was carefully pipetted into a new EP tube or 96-well plate using a pipette gun.
(4) And (3) adding 0.2 times volume of AMPure XP Beads (namely adding 8 mu l) into the supernatant obtained in the step (3), blowing up and down by using a pipette gun to fully mix the amplification product with the magnetic Beads, and standing for 2min at room temperature.
(5) And (4) after the step (4) is finished, adsorbing the magnetic beads by using a magnetic frame until the solution is clarified, carefully absorbing the supernatant by using a pipette gun, and keeping the magnetic beads.
(6) And (5) after the step (5) is finished, adding 40 mu l of 80% ethanol into the magnetic beads, suspending the magnetic beads by using a pipette gun, standing for 2min, adsorbing the magnetic beads by using a magnetic frame until the solution is clarified, sucking and removing the supernatant by using the pipette gun, and keeping the magnetic beads.
(7) After completion of step (6), 100. mu.l of 70% ethanol was added to the magnetic beads, and the magnetic beads were suspended to be washed thoroughly.
(8) And (4) after the step (7) is finished, adsorbing the magnetic beads by using a magnetic frame until the solution is clarified, and carefully removing the supernatant by using a pipette gun to avoid adsorbing the magnetic beads.
(9) And (5) placing the magnetic beads obtained in the step (8) in an oven at 50 ℃ for 5min, and evaporating the ethanol.
3. Second round PCR
The reaction was prepared as in Table 5, followed by a second round of PCR amplification. The reaction procedure is shown in Table 7.
TABLE 5
The Index-F solution was prepared by ligating Index to the 5' -ends of 24 kinds of the upstream primers shown in Table 2. In the Index-F solution, the total concentration of primers was 100. mu.M, and the molar ratios of 24 primers were equal.
The Index-R solution provides 24 downstream primers, i.e., obtained by ligating Index to the 5' -end of each of the 24 downstream primers in Table 2. In the Index-R solution, the total concentration of primers was 100. mu.M, and the molar ratios of 24 primers were equal.
The purpose of Index is to add a unique sample tag to the amplification product of each blood sample to distinguish the results of different samples in subsequent high throughput sequencing. Exemplary, 96 forms of Index are shown in Table 6. The Index in Index-F and the Index in Index-R may be the same or different. Thus, the 96 indexes of table 6 can achieve labeling of 9216(96 × 96) subjects.
TABLE 6
TABLE 7
4. Purification of PCR products
(1) After the step 3 is completed, adding 0.9 times volume of AMPure XP Beads (namely adding 9 mul) into the amplification product, blowing up and down by using a pipette gun to fully mix the amplification product with the magnetic Beads, and standing for 2min at room temperature.
(2) And (3) after the step (1) is finished, adsorbing the magnetic beads by using a magnetic frame until the solution is clarified, and absorbing and removing the supernatant by using a pipette gun to retain the magnetic beads.
(3) After completion of step (2), 40. mu.l of 80% ethanol was added to the magnetic beads, and the magnetic beads were suspended with a pipette.
(4) And (4) after the step (3) is finished, adsorbing the magnetic beads by using a magnetic frame until the solution is clarified, and absorbing and removing the supernatant by using a pipette gun to retain the magnetic beads.
(5) After the step (4) is completed, 100 μ l of 70% ethanol is added to the magnetic beads, the magnetic beads are suspended for sufficient washing, the magnetic beads are adsorbed by a magnetic rack until the solution is clarified, and the supernatant is carefully removed by using a pipette to avoid adsorbing the magnetic beads.
(6) And (5) after the step (5) is finished, placing the magnetic beads in an oven at 50 ℃ for 5min, and quickly evaporating the ethanol.
(7) And (4) after the step (6) is finished, adding 20 mul of Tris-HCl buffer solution into the magnetic beads, fully suspending the magnetic beads, standing at room temperature for 2min, adsorbing the magnetic beads by using a magnetic frame, and sucking the supernatant to obtain the DNA solution.
(8) The DNA solution was taken and the DNA concentration was measured.
Thirdly, high-throughput sequencing and obtaining genotype sequencing and data analysis of the 24 SNPs
And (3) sequentially performing the first step and the second step on each blood sample respectively.
And then mixing the products of the second step of each blood sample, and carrying out high-throughput sequencing to obtain the genotypes of the 24 SNP sites of the blood samples. High throughput sequencing may employ the sequencing platform of Illumina, e.g., Nextseq 500, Hiseq X10, Novaseq6000, or the like.
Example 4 genotyping of multiple subjects to verify uniqueness using the method of example 3
In order to verify the uniqueness of the genotypes of the 24 SNP sites in the population, 100 healthy examinees (all Han nationalities) were genotyped for the 24 SNP sites by adopting a Novaseq6000 sequencing platform according to the method of the embodiment 3.
All subjects obtained genotypes at 24 SNP sites. The results show that all individuals have unique genotyping results. The results of the genotype test of some of the subjects are shown in Table 8.
TABLE 8
| rs number | Person to be tested 1 | Person to be tested 2 | Person to be tested 3 | Test person 4 | Test subject 5 | Person under test 6 | Test person 7 | Person to be tested 8 |
| rs10875358 | TT | TT | TT | GT | GT | GG | GG | GG |
| rs297482 | CC | CT | CT | CC | TT | CC | CT | TT |
| rs62322761 | GG | AA | GG | GG | AA | AA | AG | AA |
| rs12509540 | GG | AG | AG | AG | GG | AG | AA | AA |
| rs284735 | CC | GG | GG | CC | GG | CC | CG | CC |
| rs9488534 | AA | AA | AG | AG | AG | GG | AG | AA |
| rs2470937 | TT | TT | AT | TT | AT | AT | AA | TT |
| rs10098590 | GG | CG | CC | GG | CG | CG | GG | GG |
| rs4512444 | AA | AA | AC | AA | CC | CC | CC | CC |
| rs6602770 | CT | TT | TT | CC | CT | CT | CC | CC |
| rs1904826 | TT | TT | TT | TT | AT | AT | TT | AT |
| rs11050532 | CG | GG | CG | GG | CG | CC | GG | CG |
| rs1932870 | CT | TT | CT | CT | CC | CC | CC | CC |
| rs12877845 | CC | CT | CC | TT | CC | CT | CT | TT |
| rs61266056 | AG | GG | AA | AA | AG | AA | AA | GG |
| rs935335 | CC | CT | CT | CT | CC | CT | TT | CT |
| rs2415255 | AA | AA | AG | AG | GG | AA | AA | AA |
| rs28791774 | CC | AA | AA | AA | CC | AC | AA | AC |
| rs9914679 | TT | TT | CT | TT | TT | CC | CC | TT |
| rs1893397 | CG | CC | GG | GG | CC | CG | CG | GG |
| rs71220063 | CT | CT | CC | TT | CC | CC | TT | CC |
| rs4815110 | GG | CC | CC | CC | CG | CG | CC | CC |
| rs78970726 | AA | GG | AA | GG | GG | AA | AA | GG |
| rs2004509 | GG | AA | AG | AA | GG | AG | AA | AA |
The subsequent inventors also detected a larger sample size, all obtained genotypes of 24 SNP sites, and all could realize individual identification.
Example 5 preparation of Gene identity card
Coding rule of gene identity card
The coding of the gene identity card of the invention can have various modes, such as arranging the SNP locus number (such as the SNP locus number 1, the number is 01) and the locus genotype (such as the A/T type) according to a certain rule, thereby forming a character string consisting of serial numbers and letters, and the character string is the gene identity card of an individual. Other reasonable encoding schemes may be used, and the present invention is not limited in this regard.
The coding of the gene identity card is formed by arranging 24 selected SNP genotypes according to a certain rule, wherein the specific rule is that the arrangement of the SNP genotypes is arranged from small to large according to the chromosome number of an SNP source, and the genotypes of two SNPs derived from the same chromosome are arranged from small to large according to the position information of the SNP chromosome, namely the coding is carried out according to the sequence of the SNPs in the table 1 of the embodiment 1 of the invention. The genotype of the SNP sites is arranged according to the sequence of A, C, G, T, for example, the genotype is TC and is coded as CT, and the genotype is TA and is coded as AT.
The gene identity card of the invention encoded in the above manner is composed of 48 alphabetic bars, and can be aligned for identity by Clustal Omega sequence alignment software (https:// www.ebi.ac.uk/Tools/msa/clustalo /) (see FIG. 2 for example).
Secondly, manufacturing the two-dimensional code containing the SNP locus genotype information
And (2) making the gene identity card number (a character string consisting of a string A, C, G, T) coded according to the coding mode of the step one into a two-dimensional code (as shown in an exemplary figure 1) by using common two-dimensional code generation software, and reading the gene identity card by using a mobile phone two-dimensional code scanning program.
Thirdly, preparation of the gene identity card of the invention
After obtaining a sample of an individual, extracting DNA of the sample, typing the SNP in the table 1 of the invention by a method of Sanger sequencing, KASP, high-throughput sequencing and the like to obtain a typing result of each SNP, and coding according to the coding rule of the step one to obtain DNA identity card codes of the individual, wherein the codes can be stored in a special chip, the chip can be placed in an entity card type identity card, and the DNA identity card codes in the chip can be read by scanning the chip. The two-dimensional code generator can also be used for generating individual unique DNA identity card two-dimensional codes, the two-dimensional codes are printed on the surface of the entity card type identity card, and the two-dimensional codes are scanned through a common two-dimensional code scanning program of a mobile phone, so that the individual DNA identity card codes can be read.
The specific pattern of the DNA identity card is not limited in the present invention as long as the combination of genotype information of the SNP sites listed in Table 1 is included.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Beijing Chin medical laboratory Co., Ltd
<120> a genetic marker combination and special primer combination for individual identification
<130> GNCYX202556
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<210> 11
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cctacacgac gctcttccga tctaagttaa caaaagaagt gc 42
<210> 12
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggagttcctt ggcacccgag aattccactt ctgatccatg aacatgag 48
<210> 13
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cctacacgac gctcttccga tcttaaggtg gtgagcaatg agg 43
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<211> 47
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<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggagttcctt ggcacccgag aattccaacc accacaagta cttccac 47
<210> 15
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cctacacgac gctcttccga tcttggaaat gggccttcca aag 43
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<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggagttcctt ggcacccgag aattccaata tccaaccagg caatccc 47
<210> 17
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cctacacgac gctcttccga tctacagata ggagctgatg gag 43
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
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<210> 19
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cctacacgac gctcttccga tctgaatcac ttgaacctgg gag 43
<210> 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
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<210> 22
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ggagttcctt ggcacccgag aattccaact cgggacctgg gttattg 47
<210> 23
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cctacacgac gctcttccga tctggaaata gcgccacctt ttg 43
<210> 24
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ggagttcctt ggcacccgag aattccaacg agtctccttt tttaccc 47
<210> 25
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cctacacgac gctcttccga tctcaattgt gagcatttat tagg 44
<210> 26
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ggagttcctt ggcacccgag aattccaggg cagtatattg attgctt 47
<210> 27
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
cctacacgac gctcttccga tctaatccca gctacttgag agg 43
<210> 28
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ggagttcctt ggcacccgag aattccatgc aatggtgtaa tctcggc 47
<210> 29
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
cctacacgac gctcttccga tctcaataac tccccattcc ctg 43
<210> 30
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ggagttcctt ggcacccgag aattccagag tagtcaaatt catggag 47
<210> 31
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cctacacgac gctcttccga tctatggtgg catgtgtctg tag 43
<210> 32
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ggagttcctt ggcacccgag aattccaatt acctcactgc agccttg 47
<210> 33
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cctacacgac gctcttccga tctctcccaa agtgggatta cag 43
<210> 34
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ggagttcctt ggcacccgag aattccagag accttgtcaa ggaatag 47
<210> 35
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cctacacgac gctcttccga tctccaaatg gtcatgaatg ga 42
<210> 36
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ggagttcctt ggcacccgag aattccacgg ctccatttga tgaatcc 47
<210> 37
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
cctacacgac gctcttccga tcttccccat taagctggtg aac 43
<210> 38
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ggagttcctt ggcacccgag aattccagat agatttccta cagtttc 47
<210> 39
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
cctacacgac gctcttccga tctcagcaaa ctaggaaggg aac 43
<210> 40
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ggagttcctt ggcacccgag aattccatcc catacaaagt caagcag 47
<210> 41
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
cctacacgac gctcttccga tcttatggtt tggctctgtg tcc 43
<210> 42
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggagttcctt ggcacccgag aattccaatg atccaatcac ctcccac 47
<210> 43
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
cctacacgac gctcttccga tctagtacag ctctcttgtt ggg 43
<210> 44
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
ggagttcctt ggcacccgag aattccatct ctcctaggaa ctgcaag 47
<210> 45
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
cctacacgac gctcttccga tctacagagc aagactccat ctc 43
<210> 46
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ggagttcctt ggcacccgag aattccaatg tgggtttatt tctgggc 47
<210> 47
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cctacacgac gctcttccga tcttcataac tcactgccac ctc 43
<210> 48
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ggagttcctt ggcacccgag aattccacct gtagtcctag ctacttg 47
Claims (10)
1. The application of the substance for detecting 24 SNPs in the preparation of the kit; the use of the kit is for identifying human individuals; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
2. Use of a substance for detecting 24 SNPs in human individual identification; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
3. The specific primer group is a specific primer group A or a specific primer group B;
the specific primer group A consists of 48 primers; the 48 primers are sequentially shown as a sequence 1 to a sequence 48 in a sequence table;
the specific primer group B consists of 48 primers; the 48 primers are obtained by adding sample labels at the 5' ends of the 48 primers of the specific primer group A.
4. The use of the specific primer set according to claim 3, wherein the specific primer set is (a), (b) or (c):
(a) the specific primer group is applied to identifying the genotypes of the 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509;
(b) the application of the specific primer group in the preparation of the kit; the use of the kit is for identifying human individuals;
(c) the specific primer group is applied to human individual recognition.
5. A method of personal identification of a person, comprising the steps of:
(1) detecting the genotype of a human to be tested based on 24 SNPs to obtain the corresponding 24 genotype information of the 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509;
(2) the human subject was identified by the genotype information of the 24 SNPs.
6. A method of personal identification of a person, comprising the steps of:
(1) detecting the genotype of a human to be tested based on 24 SNPs to obtain the corresponding 24 genotype information of the 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509;
(2) and making the 24 genotype information into a gene identity card, and identifying the testee through the gene identity card.
7. A genetic marker combination consisting of 24 SNPs; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
8. A gene identity card which records the genotype information of 24 SNPs of a human subject; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
9. A two-dimensional code which can read genotype information of 24 SNPs of a human subject; the 24 SNPs are as follows: rs10875358, rs297482, rs62322761, rs12509540, rs284735, rs9488534, rs2470937, rs10098590, rs4512444, rs6602770, rs1904826, rs11050532, rs1932870, rs12877845, rs61266056, rs935335, rs2415255, rs28791774, rs9914679, rs1893397, rs71220063, rs4815110, rs78970726, rs 2004509.
10. Use of the genetic marker combination of claim 7, the gene identification card of claim 8, or the two-dimensional code of claim 9 for identification of individual human.
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| CN113564266A (en) * | 2021-09-24 | 2021-10-29 | 上海仁东医学检验所有限公司 | SNP typing genetic marker combination, detection kit and application |
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