CN110863056A - Method, reagent and application for accurately typing human DNA - Google Patents

Method, reagent and application for accurately typing human DNA Download PDF

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CN110863056A
CN110863056A CN201810982139.9A CN201810982139A CN110863056A CN 110863056 A CN110863056 A CN 110863056A CN 201810982139 A CN201810982139 A CN 201810982139A CN 110863056 A CN110863056 A CN 110863056A
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李生斌
苗鑫垚
常辽
于慧云
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Shenzhen Huada Forensic Science And Technology Co Ltd
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Abstract

The application discloses a method, a reagent and application for accurately typing human DNA. The accurate human DNA typing method comprises the steps of simultaneously detecting short tandem repeat sequences, polynucleotide polymorphic sites covering a whole genome, a mitochondrial DNA hypervariable region I, a mitochondrial DNA hypervariable region II and an Amel enamel gene to obtain accurate typing and base sequences of each site, so that accurate typing of human DNA is realized. The method has high resolution, can carry out high-efficiency and accurate individual identification, has extremely high individual identification power for difficult and difficult material detection and highly degraded material detection, has a non-father exclusion rate of more than 99.999999 percent in paternity test, and has high accuracy; the compatibility is good, the existing individual recognition detection kit can be covered, and the human genome data can be combined to be used for analyzing ethnic groups in different regions or countries; in addition, the method has high material detection adaptability, and can detect human DNA extracted by various experimental methods.

Description

Method, reagent and application for accurately typing human DNA
Technical Field
The application relates to the field of human DNA typing detection methods, in particular to a method, a reagent and application for accurately typing human DNA.
Background
Short Tandem Repeat (STR), also known as microsatellite DNA or simple repeat (SSR), is the most widely used genetic marker of length polymorphism in forensic physical evidence identification at present, STR has short repeat units of only 2-6bp, and its length polymorphism is derived from individual differences in the copy number of the repeat units.
On the human genome scale, Single Nucleotide Polymorphisms (SNPs) are formed if any base mutation makes two bases occur at a specific nucleotide position, the frequency of the least one of which in the population is not less than 1%. Because of the abundant SNP content and the high genetic stability, the application of the SNP in the aspect of forensic physical evidence inspection draws high attention, and the SNPs are also considered as third-generation genetic markers following STR.
With the discovery of sequencing technology, mitochondrial DNA (mtDNA) has become a routine protocol; the focus of forensic mtDNA sequence analysis is the hypervariable regions (HVRs) I and II. When the amount of nuclear DNA is insufficient to perform typing, the mitochondrial DNA analysis is also the only technique available, and for example, hair shafts, bones, teeth or other severely degraded materials in biological materials depend on the analysis of mitochondrial DNA.
Human genetics is an important foundation for forensic physical evidence. Human inheritance and variation allow each individual to have genetic information similar to that of his parents and progeny, but each individual has its own genetic characteristics. Forensic physical evidence is a research on applying the genetic rules to individual identification and parental authentication practices, and provides accurate and effective scientific evidence for case detection and judgment.
The current individual identification technologies mainly include one generation STR detection technology and the next generation sequencing (abbreviated NGS) based detection technology. Among them, in the first generation STR detection technology, STR is a polymorphic locus in human genome, which is composed of 2-6 base pairs as core sequence, and is arranged in series and repeat, STR locus length is generally between 100 and 300bp, STR length difference between individuals constitutes polymorphism, and in gene transmission, it follows Mendel's law of heredity, and has been widely applied to individual identification and paternity test in forensic medicine. At present, the market generally comprises about 18 STR loci detection kits. Among the NGS-based detection technologies, illumina fgx, which is a similar product, performs paternity test and individual identification based on STR and SNP (single nucleotide polymorphism) sites.
The existing first generation STR detection technology in the market usually adopts capillary electrophoresis detection, about 20 sites can be detected, the number of the detected sites is small, and the difficult and complicated relation judgment is difficult to determine; in addition, the first generation STR detection technology can only judge the typing result according to the allele length, and has low detection degree on sample genetic variation and allele loss. The NGS-based detection technology, such as Illumina FGx, has certain defects for maternal traceability and database construction and investigation based on sex crime.
Therefore, there is a need to develop a new method for typing human DNA more precisely so as to identify individuals more accurately and efficiently.
Disclosure of Invention
The purpose of the application is to provide a novel method, reagent and application for accurately typing human DNA.
The application specifically adopts the following technical scheme:
one aspect of the application discloses a method for accurately typing human DNA, which comprises the steps of simultaneously detecting short tandem repeat sequences, polynucleotide polymorphic sites covering a whole genome, a mitochondrial DNA hypervariable region I, a mitochondrial DNA hypervariable region II and an Amel enamel gene to obtain accurate typing and base sequences of each site, thereby realizing accurate typing of the human DNA.
The method for accurately typing human DNA simultaneously detects a plurality of short tandem repeat sequences, a plurality of polynucleotide polymorphic sites covering the whole genome, two mitochondrial DNA hypervariable regions and an Amel enamel gene, and realizes accurate typing based on the detection information of the sites.
Preferably, the short tandem repeat sequence comprises 143 STRs and the polymorphic site of the polynucleotide covering the whole genome comprises 188 SNPs. In one implementation of the present application, specific 143 STRs and 188 SNPs are shown in table 1.
The DNA precise typing method of the application simultaneously comprises 143 STRs, 188 SNPs, two sections of mitochondrial hypervariable regions and Amel enamel genes, has high resolution, good compatibility, high individual identification capacity, high material detection adaptability and high accuracy, and can be applied to various fields needing DNA precise typing.
Preferably, the method for accurately typing human DNA further comprises the steps of performing PCR amplification on 143 STRs, 188 SNPs, a mitochondrial DNA hypervariable region I, a mitochondrial DNA hypervariable region II and an Amel enamel gene by using a primer group, and sequencing the PCR amplification product to obtain accurate typing and base sequences of each site.
It should be noted that, the sequencing after PCR amplification of each detection site or target is a detection method that is currently and conventionally used, and does not exclude that other means can be used for detection or sequencing, and is not specifically limited herein.
Preferably, in the method for accurately typing human DNA of the present application, the primer set includes primers having sequences shown in SEQ ID No.1 to SEQ ID No.668, wherein the sequences shown in SEQ ID No.1 and SEQ ID No.2 are a pair of primers, the sequences shown in SEQ ID No.3 and SEQ ID No.4 are a pair of primers, the sequences shown in SEQ ID No.5 and SEQ ID No.6 are a pair of primers, and so on.
It should be noted that, in the PCR amplification, a pair of upstream and downstream primers are usually used to amplify the target fragment, and the primers of the sequences shown in SEQ ID No.1 to SEQ ID No.668 of the present application are also paired pairwise, that is, the sequences shown in SEQ ID No.1 and SEQ ID No.2 are a pair of primers, the sequences shown in SEQ ID No.3 and SEQ ID No.4 are a pair of primers, and so on, until the sequences shown in SEQ ID No.667 and SEQ ID No.668 are a pair of primers; thus, the primer set of the present application actually comprises 334 primer pairs, and these 334 primer pairs can cover 143 STRs, 188 SNPs, two mitochondrial hypervariable regions and Amel enamel genes defined in the present application, and in principle one primer pair amplifies one site, but there are also some amplified fragments of the primer pairs that can cover two or more sites. It is understood that 334 primer pairs of the present application cover 143 STRs, 188 SNPs, two segments of mitochondrial hypervariable regions and Amel enamel genes, and some primer pairs may be selected for human DNA typing according to different use requirements, which is not specifically limited herein.
The application also discloses application of the human DNA precise typing method in paternity test, pedigree construction, pedigree tracing, bank building and investigation based on sex crime, full sibling relationship test, half sibling relationship test, difficult material DNA typing detection or high degradation material DNA typing detection.
It should be noted that the accurate human DNA typing method of the present application can accurately and efficiently identify individuals, and thus can be completely used for paternity test, family tree construction or traceability, identification of full-sib or half-sib relationships, and the like. Moreover, the method of the application covers two sections of mitochondrial hypervariable regions, so the method is also suitable for typing detection of DNA of difficult and complicated test materials or typing detection of DNA of highly degraded test materials. Wherein, the paternity test comprises standard diad and triplet test. It will be appreciated that the method of the present application is in principle applicable to a variety of application areas based on efficient, accurate individual identification.
The application also discloses a reagent for accurate typing detection of human DNA, which comprises primers of sequences shown in SEQ ID NO.1 to SEQ ID NO.668, wherein the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are a pair of primers, the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 are a pair of primers, the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 are a pair of primers, and the like.
It should be noted that the reagent for accurately typing human DNA is mainly used for performing PCR amplification on a target fragment, and then detecting a PCR amplification product, such as high-throughput sequencing, to realize accurate typing of human DNA.
It is understood that the reagents of the present application are equally applicable to the various fields to which the methods of the present application for the precision typing of human DNA are applicable, and are not specifically limited herein.
Still another aspect of this application discloses a kit for accurate typing detection of human DNA, which includes the reagent of this application.
Preferably, the kit of the present application further comprises at least one of a DNA extraction reagent, a PCR amplification reagent, a sequencing library construction reagent, and a nucleic acid purification reagent.
It is understood that the reagents of the present application can be conveniently developed into a kit, wherein the DNA extraction reagent, the PCR amplification reagent, the sequencing library construction reagent and the nucleic acid purification reagent can also be selectively put into the kit for convenient use. Of course, the reagents for DNA extraction, PCR amplification, sequencing library construction and nucleic acid purification may be reagents conventionally used in laboratories, and are not particularly limited.
The beneficial effect of this application lies in:
the accurate human DNA typing method combines a plurality of short tandem repeat sequences, a polynucleotide polymorphic site covering a whole genome, two mitochondrial DNA hypervariable regions and an Amel enamel gene for detection, has high resolution, can carry out high-efficiency and accurate individual identification, has extremely high individual identification capability on difficult and difficult materials and highly degraded materials, has a non-father exclusion rate of more than 99.999999 percent in paternity identification, and has high accuracy; the compatibility is good, the existing individual recognition detection kit can be covered, and the human genome data can be combined to be suitable for ethnic group analysis of different regions or countries; in addition, the method has high material detection adaptability, and can detect and precisely classify the human DNA extracted by various experimental methods.
Drawings
FIG. 1 is a plot of the depth of site at which three unrelated samples were examined in an example of the present application.
Detailed Description
The existing individual identification technology, such as a generation STR detection technology and an NGS-based detection technology, has certain defects, and is difficult to realize accurate typing of human DNA. For this purpose, the application creatively provides that the short tandem repeat sequences, the polynucleotide polymorphic sites covering the whole genome, the mitochondrial DNA hypervariable region I and the mitochondrial DNA hypervariable region II are simultaneously detected, so as to carry out the accurate typing of the human DNA.
In an implementation manner of the application, a typing method capable of detecting and analyzing 143 STRs, 188 SNPs, two sections of mitochondrial hypervariable regions and Amel enamel genes is provided, the method combines human genome data, selects yellow, white and black characteristic loci, and can be applied to standard diad and triplet paternity identification, pedigree construction or tracing, database construction and investigation based on sex crimes, identification of homomorphic and hemimorphic relations and typing detection of difficult and complicated degradation samples. Wherein, each detection site is obtained by screening 143 STRs, 188 SNPs, two sections of mitochondrial hypervariable regions and Amel enamel genes for detecting sex finally by the statistical research of the polymorphism and mutation rate of the STR sites, the SNP sites and the mitochondrial hypervariable regions and combining the STR sites required by public security library construction.
The method for accurate typing of human DNA of the present application:
(1) the resolution ratio is high, and the preferable scheme of the application simultaneously comprises 143 STRs, 188 SNPs, two sections of mitochondrial hypervariable regions and Amel enamel genes, and can be applied to standard diads and triplets paternity identification, family tree construction or traceability, database construction and investigation based on sex crimes, identification of full-sib and half-sib relations, and typing detection of difficult degradation samples.
(2) The compatibility is good, the kit can cover most of the first-generation and second-generation kits on the market, and is suitable for ethnic groups of different regions and countries such as yellow race, white race, black race and the like by combining human genome data.
(3) The individual recognition capacity is high, in the preferred scheme of the application, 143 STRs, 188 SNPs, two sections of mitochondrial hypervariable regions and Amel enamel genes are simultaneously included, the number of coverage sites is far beyond the number of the existing first-generation and second-generation kits, and the individual recognition capacity for difficult and complicated test materials and highly degraded test materials is extremely high.
(4) The method can carry out accurate typing detection on human DNA from different sources obtained by various test methods, such as human DNA extracted by a chelex100 method, a magnetic bead method, a resin purification method and the like, and samples such as blood, blood filter paper, sperm stain, oral swab and the like.
(5) The accuracy is high, and when the method is used for paternity test, the number of sites is large and the sites cover the whole genome and the mitochondrial group, so that the non-paternal exclusion rate is greater than 99.999999%.
(6) In one implementation of the present application, the methods of the present application are combined with high throughput sequencing, utilizing second generation sequencing and multiplex amplification techniques to improve site data uniformity; and high-throughput sequencing has obvious advantages in terms of throughput and cost, and 334 sites can be amplified by using a composite amplification technology. The second-generation sequencing has certain application in forensic material evidence due to the advantages of high flux, low cost, time saving and the like, and has wider application prospect along with the continuous reduction of sequencing cost.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
First, site selection and primer design
In the example, 143 STRs, 188 SNPs, two sections of mitochondrial hypervariable regions and Amel enamel genes are finally screened by statistically researching STR loci, SNP loci and polymorphism and mutation rate of mitochondrial hypervariable regions and simultaneously combining the required STR loci of a public security library.
Specific primers are respectively designed on the flanks of the sequences of 143 STR, 188 SNP, two sections of mitochondrial hypervariable regions and Amel enamel genes. Primer design adopts PrimerPrimier5 and Oligo7 software, the annealing temperature of each primer is about 58 ℃, primer dimer or other non-specific products caused by mismatch cannot be generated, and the length of the amplification product is between 150 and 400 bp. The sequences of the final primers of this example are shown in Table 1.
TABLE 1 detection sites and primers
Figure BDA0001778828600000061
Figure BDA0001778828600000071
Figure BDA0001778828600000081
Figure BDA0001778828600000091
Figure BDA0001778828600000101
Figure BDA0001778828600000111
Figure BDA0001778828600000121
Figure BDA0001778828600000131
Figure BDA0001778828600000141
Figure BDA0001778828600000151
Figure BDA0001778828600000161
Figure BDA0001778828600000171
Figure BDA0001778828600000181
Figure BDA0001778828600000191
Figure BDA0001778828600000201
Figure BDA0001778828600000211
In table 1, HVR I and HVR II are mitochondrial DNA hypervariable region I and mitochondrial DNA hypervariable region II, respectively; the naming rule of the SNP loci is that the locus name "rs" is the beginning of the SNP loci, and M479, L298, P202 and P256 are Y chromosome haplotypes except the SNP loci beginning at "rs"; the nomenclature of STRs is that STRs located in the coding region of proteins, including the genes of the intron regions, are named using the coding strand, which applies to VWA (GenBank: M25716), TPOX (Gc: nBank: M68651) and CSFIPO (CenBank: X14720), repetitive sequences unrelated to the protein-coding gene, such as the D # S # locus, such as D18S51 (GenBank; L18333), D21Sll (GenBank; M84567), although other nomenclature is included in Table 1. The STR name and the name at the beginning of "rs" of the SNP used in this example can be searched from databases such as NCBI to the corresponding position of the genome, and other names are also publicly used names.
In table 1, locus DYS389i and DYS389 ii primer sequences are identical, DYF387S1a and DYF387S1b primer sequences are identical, DYS385a and DYS385b primer sequences are identical, DYS527a and DYS527b primer sequences are identical, DYS526a and DYS526b primer sequences are identical, and dysf 404S1a and dysf 404S1b primer sequences are identical, and in multiplex amplification, a pair of primers can amplify two loci at the locus where the primer sequences are identical.
Second, DNA extraction
This example was tested using six random unrelated individuals, three males and three females, and two negative controls. Samples of six random unrelated individuals were collected from volunteer samples from six different families, and nuclease-free water was used for two negative controls. The six random unrelated individuals comprise 60 autosomal STRs, 53Y-STRs, 31X-STRs, 122 autosomal SNPs, 35Y-SNPs, 34X-SNPs, 628bp mitochondrial hypervariable regions and Amel enamel genes, so that the total number of male loci is 144 STRs, 188 SNPs and two mitochondrial hypervariable regions, and the total number of female loci is 91 STRs, 154 SNPs, two mitochondrial hypervariable regions and Amel enamel genes.
In the embodiment, the rapid DNA extraction kit of Tiangen biochemical technology is adopted to extract different types of DNA samples, a Qubit fluorescence quantitative method is used for detecting the DNA concentration, and the DNA samples with higher or lower concentration are diluted and concentrated. Finally, the concentration of all DNA samples in the embodiment is in the range of 3-10 ng, which can meet the requirements of subsequent experiments.
Third, PCR amplification
The PCR amplification is carried out on the extracted DNA sample by adopting the composite amplification technology of the embodiment, and the reaction system comprises: reaction buffer 10. mu.L, primer mixture 2.5. mu.L, Taq DNA polymerase 1. mu. L, DNA sample 2.5. mu. L, ddH2O9. mu.L, 25. mu.L in total.
Wherein, the reaction buffer system comprises: 10mM DMSO, 50mM KCl, 10mM Tris-HCl (pH8.3, 25 ℃ C.), 2.0mM MgCl20.1mg/ml BSA (bovine serum albumin) and dNTPs each having a deoxyribonucleotide concentration of 0.2 mM.
The primer mixture is an equal ratio mixture of 334 pairs of primers in table 1, and all the primer pairs react in one system.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; then 30 cycles were entered: denaturation at 95 ℃ for 20 seconds, annealing at 58 ℃ for 1 minute, and extension at 72 ℃ for 20 seconds; after the circulation is finished, extending for 10 minutes at 72 ℃; storing at 4 ℃.
Fourth, library construction and sequencing
And amplifying the amplification system according to the program to obtain an amplification product mixture at each site, carrying out Qubit fluorescence quantification on the composite amplification product, and taking out 50ng of the amplification product to construct a second-generation sequencing library of the Huada gene BGISEQ-500 platform.
The used library building kit and sequencing kit are kits matched with a BGISEQ-500 platform.
The quantified composite amplification product is supplemented to 40 mu L by water, and then the end repair Buffer Mix and the end repair Enzyme Mix are added for end repair and A tail adding treatment. The reaction volume is 50 mu L/tube, and the reaction condition is that the hot cover is 110 ℃; 30min at 37 ℃; 15min at 65 ℃; storing at 4 ℃.
After the reaction is finished, adding 5 mu of LAdaptor Mix into the PCR tube, blowing, beating and uniformly mixing; subsequently, 20.4. mu.L of linker ligation Buffer was added, and 4.6. mu.L of Enzyme Mix was ligated to the linker. The reaction volume is 80 mu L/tube, and the reaction condition is that the hot cover is 110 ℃; 60min at 20 ℃; storing at 4 ℃.
After the reaction, the PCR tube was taken out, and 20. mu.L of nuclease-free water was added thereto to make up to 100. mu.L. Purify with 1-fold volume, i.e. 100. mu.L, Ampure XP beads, and after purification, re-dissolve with 46. mu.L water, aspirate 44. mu.L for subsequent experiments.
To the ligated 44. mu.L of the purified product, Pre-PCR Enzyme 100. mu. L, Pre-PCR Primer Mix 12. mu.L and nuclease-free water 44. mu.L were added in a total volume of 200. mu.L, and the mixture was mixed and dispensed into two PCR tubes for PCR reaction. The reaction volume is 100 mul/tube, and the reaction conditions are 95 ℃ for 3 minutes; then 6 cycles were entered: denaturation at 98 ℃ for 20 seconds, annealing at 60 ℃ for 15 seconds, and extension at 72 ℃ for 15 seconds; after the circulation is finished, extending for 10 minutes at 72 ℃; storing at 4 ℃.
And each PCR tube is purified by adopting 1.5 times of volume, namely 300 mu L of Ampure XP magnetic beads, and is finally dissolved in 40 mu L of water after purification is finished, all the PCR tubes are absorbed, and quantification is carried out by a Qubit fluorescence quantification method. The quantitative analysis result shows that the obtained product can meet the use requirement of the second-generation sequencing of the BGISEQ-500 platform; therefore, the constructed library sample is subjected to BGISEQ-500 platform second-generation sequencing.
Fifth, testing the detection rate of the locus
In this example, the sequencing results of six random unrelated individuals and two negative controls were analyzed, and the site detection rates were counted, the results are shown in Table 2.
TABLE 2 results of site detection rates
Figure BDA0001778828600000231
The results in Table 2 show that the method of this example has a site detection rate of at least 99% for all three male samples and a detection rate of up to 100% for all three female samples, while no detection is found for the negative control sample, indicating that the method of this example has a high site detection rate and can be used for DNA precise typing analysis.
Sixthly, verifying the accuracy of the locus
Since the number of sites contained in the conventional one-generation kit is limited, the human genome standard 9948 is selected in the present example, and a plurality of one-generation sequencing kits are used to obtain the typing results, which are compared with the sequencing typing results in the present example. Specifically, the first-generation sequencing kit used in this example: the human DNA typing kit (Yanhuang, cat # FGI1905) and the human DNA typing kit (Yanhuang Y, cat # FGI1907) are capillary electrophoresis kits.
Aspirate 2.5. mu.L of human genome standard 9948 into the PCR reaction system.
The PCR reaction system comprises: reaction buffer 10. mu.L, primer mixture 2.5. mu.L, Taq DNA polymerase 1. mu. L, DNA sample 2.5. mu. L, ddH2O9. mu.L, 25. mu.L in total.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; then 30 cycles were entered: denaturation at 95 ℃ for 20 seconds, annealing at 58 ℃ for 1 minute, and extension at 72 ℃ for 20 seconds; after the circulation is finished, extending for 10 minutes at 72 ℃; storing at 4 ℃.
After the PCR amplification reaction is finished, taking out the PCR tube, and purifying by using XP magnetic beads with 2 times of the volume of the PCR product, namely adding 100 mu L of XP magnetic beads into the PCR amplification product for purification, wherein the XP magnetic bead purification refers to an AgencurtAmpure XP-Medium kit. And establishing a high-throughput sequencing library, referring to the fourth step of library construction and sequencing in the example, performing computer sequencing, and performing filtration analysis and comparison on sequencing data to obtain the site detection information of the human genome standard 9948.
For the capillary electrophoresis kit, the human genome standard 9948 was subjected to PCR amplification according to the human DNA typing kits, i.e., yanhuang and yanhuang Y, respectively. PCR amplification productBy Applied
Figure BDA0001778828600000242
3100/3500 the genetic analyzer performs capillary electrophoresis detection. The detection result in the genetic analyzer is analyzed by Genemapper ID software to obtain the capillary electrophoresis result of the standard 9948 detected by the capillary electrophoresis kit. The PCR amplification step is specifically carried out by referring to the human DNA typing kit-usage instruction of Yanhuang cat # FGI1905 and the human DNA typing kit-usage instruction of Yanhuang Y cat # FGI 1907.
The site information of the standard 9948 based on high throughput sequencing in this example was compared with the site information of the standard 9948 detected by the capillary electrophoresis kit, and some results are shown in table 3.
TABLE 3 comparison of results of 9948 locus detection of human genome standards
Figure BDA0001778828600000241
Figure BDA0001778828600000251
The comparison result shows that the result of the 9948 locus information of the standard substance based on high-throughput sequencing is highly consistent with that of the 9948 locus information of the standard substance detected by the capillary electrophoresis kit, which indicates that the detection method of the embodiment can replace the existing capillary electrophoresis kit.
Seventhly, sample preference test
Randomly obtaining off-line data of three unrelated individual samples in the step of testing the detection rate of the five sites and the site, and removing low-quality Reads in the off-line data by adopting Adapter removal (v2.1.7); the depth of each site in the PCR system was counted and a depth profile was plotted, the results of which are shown in FIG. 1.
The results in fig. 1 show that the multiplex amplification is well balanced among the samples, and a good amplification effect can be obtained for each sample, without sample preference.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

Claims (10)

1. A method for accurately typing human DNA, comprising: the method comprises the steps of simultaneously detecting short tandem repeat sequences, polynucleotide polymorphic sites covering a whole genome, a mitochondrial DNA hypervariable region I, a mitochondrial DNA hypervariable region II and an Amel enamel gene to obtain accurate typing and base sequences of each site, thereby realizing accurate typing of human DNA.
2. The method of claim 1, wherein: the short tandem repeat sequence comprises 143 STRs, and the polynucleotide polymorphic site covering the whole genome comprises 188 SNPs.
3. The method of claim 2, wherein: 143 of the STRs include D1S1656, D1GATA113, D1S1627, D1S1677, D2S441, D2S1776, D2S1338, TPOX, D3S3045, D3S1744, D3S1358, D3S3053, D3S4529, D4S2366, D4S2364, D4S2408, FGA, D5S818, D5S2500, CSF1PO, D6S477, D6S1043, D6S474, D6S1017, SE33, D7S820, D7S3048, D1179, D8S1115, D9S 627, D9S 1249, D9S2157, D10S 1122, D10S 1438, D11S2368, D11S4463, D01, D6312S 391, D12S391, D925, D9S 1248, D2157, D9S2157, D10S 293, D293S 444, D2S 293, D2S 1258, D293, D2S 12542, D293, D187, D187, D2S 444, D187, D46, D187, D527, D187, D2S, D527, D187, D1632S, D187, D1632S, D527, D1632S, D527, D1632S, D527, D1632S 444, D1632S 1258, D1632S 1258, D1632S, D1632, DYF404S1a/B, DYS508, DYS526a/B, DYS593, DYS596, DYS617, DYS626, DYS645, GATA165B12, DXS9902, DXS8378, DXS7423, DXS7132, DXS6810, DXS6807, DXS6803, DXS6795, DXS10159, DXS10103, DXS10101, GATA31E08, GATA172D05, HPRTB, DXS 100S 74, GATA144D04, DXS10161, DXS10160, DXS10162, S10164, DXS10079, GATA31E 3575, DXS981, DXS6800, DXS9898, DXS6801, S7424, DXS101, DXS7133 and DXS 7130;
the 188 SNP sites include rs1413212, rs560681, rs1490413, rs10495407, rs891700, rs7520386, rs4847034, s1294331, rs907100, rs1109037, rs876724, rs993934, rs12997453, rs4364205, rs1355366, rs1357617, rs 64444444724, rs1872575, rs2399332, rs1979255, rs 204636361, rs 291236818, rs1554472, rs13134862, rs717302, rs338882, rs 77606, rs 04770, rs13182883, rs251934, rs727811, rs13218440, rs214955 955, rs 60955, rs 606060606060435445, rs 28120, rs 25072569, rs 3005637569, rs 72563756375637569, rs 725637569, rs 725637725637729, rs 725637725637725637729, rs 725637725637725637725637729, rs 725637729, rs 725637725637725637729, rs 725637725637729, rs 725637725637725637729, rs 725637729, rs 725637725637725637729, rs729, rs 725637725637725637725637729, rs 725637725637725637725637725637729, rs729, rs 725637725637725637729, rs 725637725637729, rs 725637729, rs 725637725637729, rs729, rs 725637729, rs 725637725637729, rs 725637725637725637725637725637725637725637729, rs729, rs 725637729, rs 725637725637725637729, rs729, rs 725637729, rs 725637725637725637725637729, rs 725637729, rs729, rs 725637729, rs729, rs, rs5906919, rs9781645, rs1874111, rs471205, rs2209420, rs5968332, rs6418330, rs5923750, rs1166756, rs2808742, rs5916781, rs6646036, rs594031, rs5932595, rs5933388, rs5931302, rs5908324, rs2128519, rs1534285, rs 76766, rs993010, rs1557054, rs1243792, rs925178, rs1977719, rs 54989825, rs 193061930674, rs7471388, rs2056688, rs 2534344978610, rs9786184, rs9786139, rs 1698129129129129129129129129129129129129129845, L298, P256, rs 20320320320320320320320320341886, rs 2595, rs 252599, rs 30017320, rs 2602, rs 021791727946, rs 417946, rs 411725017250845, rs 264746, rs 26989, rs 269826, rs 26989, rs 269825147443, rs 2698443, rs 26989 and rs 26989.
4. The method of claim 2, wherein: the method also comprises the steps of carrying out PCR amplification on 143 STRs, 188 SNPs, a mitochondrial DNA hypervariable region I, a mitochondrial DNA hypervariable region II and an Amel enamel gene by adopting a primer group, and then sequencing PCR amplification products to obtain accurate typing and base sequences of all the sites.
5. The method of claim 4, wherein: the primer group comprises primers of sequences shown by SEQ ID NO.1 to SEQ ID NO.668, wherein the sequences shown by SEQ ID NO.1 and SEQ ID NO.2 are a pair of primers, the sequences shown by SEQ ID NO.3 and SEQ ID NO.4 are a pair of primers, the sequences shown by SEQ ID NO.5 and SEQ ID NO.6 are a pair of primers, and the like.
6. Use of the method of any one of claims 1-5 for paternity testing, pedigree construction, pedigree tracing, bank construction and investigation based on sex crime, holomorphic relationship testing, hemimorphic relationship testing, DNA typing detection of difficult and problematic test materials, or DNA typing detection of highly degraded test materials.
7. A reagent for accurate typing detection of human DNA, characterized in that: the primers comprise sequences shown by SEQ ID NO.1 to SEQ ID NO.668, wherein the sequences shown by SEQ ID NO.1 and SEQ ID NO.2 are a pair of primers, the sequences shown by SEQ ID NO.3 and SEQ ID NO.4 are a pair of primers, the sequences shown by SEQ ID NO.5 and SEQ ID NO.6 are a pair of primers, and the like.
8. Use of the reagent of claim 7 in paternity testing, pedigree construction, pedigree tracing, bank construction and investigation based on sex crime, holomorphic relationship testing, hemimorphic relationship testing, DNA typing detection of difficult and complicated material or DNA typing detection of highly degraded material.
9. A kit for accurate typing detection of human DNA, which is characterized in that: comprising the reagent of claim 7.
10. The kit of claim 9, wherein: also comprises at least one of a DNA extraction reagent, a PCR amplification reagent, a sequencing library construction reagent and a nucleic acid purification reagent.
CN201810982139.9A 2018-08-27 2018-08-27 Method, reagent and application for accurately typing human DNA Pending CN110863056A (en)

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