CN110499373A - High-throughput STR typing system and kit for identifying complex genetic relationships - Google Patents

Abstract

The invention discloses a high-throughput STR typing system and a typing kit for complex genetic relationship identification, and relates to the technical field of nucleic acid in vitro detection. The STR typing system includes PCR primers for the amplification of 60, 118, or 179 linked autosomal STR loci, and the kit includes PCR primer sets, Index adapter sequences, IGT‑EM707 polymerase mix, amplification buffer enhancer NB, YF buffer B and library preparation reagents, etc. The STR typing system and its kit provided by the present invention can achieve single-tube amplification of 60, 118 or 179 linked autosomal STR loci, with good balance, high sensitivity, good specificity, and accurate typing results. It can be used to identify complex kinship relationships with different kinship relationships at the same level.

Description

High-throughput STR typing system and kit for identifying complex genetic relationships

technical field

The invention relates to the technical field of forensic detection, in particular to a high-throughput STR typing system and a typing kit for complex genetic relationship identification.

Background technique

Short tandem repeat (short tandem repeat, STR) is a special sequence composed of 2-6bp repeating units in series. The number of STR loci is large and widely distributed, accounting for about 3% of the entire genome, and its polymorphism is high. It is mainly derived from the difference in the number of core sequence repeats among individuals, which follows the Mendelian law of inheritance in the genetic process. Therefore, STR amplification detection technology is widely used in forensic individual identification, kinship identification and population genetics research. The most commonly used STR typing techniques are fluorescent labeling multiplex amplification combined with capillary electrophoresis (capillary electrophoresis, CE) typing technique and next-generation sequencing technique.

The identification of complex kinship is one of the technical problems that urgently need to be solved in the field of forensic identification. In recent years, there has been an increasing trend of such cases. Therefore, the success of identification of such cases often depends on the blood relationship and social relationship of several families, which is not only a huge challenge for forensic geneticists but also has a great impact on the vital interests of ordinary people.

In the identification of complex kinship relationships, there will be the need to identify different kinship relationships at the same level, such as half-siblings and uncles (aunts), half-siblings and grandparents, or uncles and nephews. The class relationship belongs to the second level, and the Identity by decent (IBD) value and the common ancestry coefficient (θ) value derived from Mendel's genetic law are consistent, so it is impossible to apply the existing independent genetic markers and calculation methods. Three relationships are distinguished. Some scholars have suggested that although age information and other DNA information, such as mitochondrial DNA and sex chromosome DNA, can be used to identify this kind of genetic relationship at the same level, it can be helpful to judge such problems, but in the face of the general situation of different genetic relationships at the same level, Linked autosomal genetic markers can better solve the above problems. In theoretical research, in 1998, Thompson used the recombination rate of the law of linkage inheritance to deduce the different kinship coefficients among grandparents, half-siblings, uncles and nephews. With the application of computer technology in the field of forensic evidence, in 2008 According to the different kinship coefficients obtained by Thompson, Egeland made a theoretical calculation model using a certain number of linked genetic markers.

At present, the existing technology on the identification of kinship relationship, for example: Chinese patent CN104818323B discloses a genotyping detection kit for 20 STR loci of human chromosomes 13, 18 and 21, which can realize a single tube of 20 STR loci Amplification; Chinese patent CN106906292A discloses a method for compound amplification of 22 short tandem repeat sequences and its kit, which can be used to amplify 22 STR loci and 1 sex locus. Most linked STR studies are X-STR and Y-STR genetic markers, which require special kinship before they can be applied; independently inherited autosomal STRs cannot distinguish complex kinship.

According to the characteristics of linked autosomal STRs, the present invention customizes the STR typing system for multiple PCR targeted capture sequencing. The STR typing system can amplify all STR genetic markers at one time, and the primer combination sequence used is well balanced and can To ensure that all loci in the group are detected, through next-generation sequencing and subsequent data analysis, the typing and sequence information of each STR can be obtained, which can be used to identify complex genetic relationships of different genetic relationships at the same level.

Contents of the invention

The purpose of the present invention is to provide a STR typing system and a kit thereof for identification of complex genetic relationships. Overcoming the technical problems existing in the prior art, the STR typing system and its kit can achieve single-tube amplification of 179 linked autosomal STR loci, with good balance, high sensitivity, good specificity, and easy typing. The results are accurate and can be used to identify complex genetic relationships with different genetic relationships at the same level.

On the other hand, in the course of the research, it was found that the above-mentioned 179 STR genomes can be divided into three independent groups, and the six STR locus combinations formed by the three groups of STR loci individually or in pairs can all be combined with the above-mentioned 179 STR loci. The same, similar or equivalent technical effect of the typing system composed of loci.

based on above:

First, the present invention provides a STR typing system for complex genetic relationship identification, said STR typing system includes PCR primer combination 1 for amplifying 60 linked autosomal STR loci;

The 60 linked autosomal STR loci, their corresponding physical positions, chromosome partitions and genetic distances on the reference genome Hg38 are:

The PCR primer combination 1 includes a forward primer set and a reverse primer set; the forward primer set is:

The reverse primer set is:

Second, the present invention provides a STR typing system for complex genetic relationship identification, said STR typing system includes PCR primer combination 2 for amplifying 58 linked autosomal STR loci;

The 58 linked autosomal STR loci, their corresponding physical positions, chromosome partitions and genetic distances on the reference genome Hg38 are:

The PCR primer combination 2 includes a forward primer set and a reverse primer set; the forward primer set is:

The reverse primer set is:

Third, the present invention provides a STR typing system for complex genetic relationship identification, said STR typing system includes PCR primer combination 3 for amplifying 61 linked autosomal STR loci;

The 61 linked autosomal STR loci, corresponding physical positions, chromosome divisions and genetic distances on the reference genome Hg38 are:

The PCR primer combination 3 includes a forward primer set and a reverse primer set; the forward primer set is:

The reverse primer set is:

Fourth, the present invention provides a STR typing system for identification of complex kinship, said STR typing system includes PCR primer combination 4 for amplifying 118 linked autosomal STR loci; said The 118 linked autosomal STR loci are STR loci whose sequence numbers are 1-118 in the description of the present invention; the PCR primer combination 4 is used to amplify the sequence number of the STR site in the description of the present invention is 1 Forward and reverse primer sets for the STR locus of -118, consisting specifically of primer sets 1 and 2.

Fifth, the present invention provides a STR typing system for complex genetic relationship identification, said STR typing system includes PCR primer combination 5 for amplifying 121 linked autosomal STR loci; said The 121 linked autosomal STR loci are the STR loci with the STR site numbers 1-60 and 119-179 in the description of the present invention; the PCR primer combination 5 is used to amplify the STR positions in the description of the present invention. The forward and reverse primer combinations of the STR loci with the point numbers 1-60 and 119-179 are specifically composed of primer combinations 1 and 3.

Sixth, the present invention provides a STR typing system for complex genetic relationship identification, the STR typing system includes PCR primer combination 6 for amplifying 119 linked autosomal STR loci; The 119 linked autosomal STR loci are STR loci whose sequence numbers are 61-179 in the description of the present invention; the PCR primer combination 6 is used to amplify the sequence number of the STR site in the description of the present invention is 61 Forward and reverse primer sets for the STR locus of -179, consisting specifically of primer sets 2 and 3.

Seventh, the present invention provides a kind of STR typing system that is used for identification of complex kinship relationship, described STR typing system comprises PCR primer combination 7 for amplifying 179 linked autosomal STR loci; The 179 linked autosomal STR loci are the STR loci with the sequence numbers of 1-179 in the description of the present invention; the PCR primer combination 7 is used to amplify the sequence number of the STR site in the description of the present invention is 1 Forward and reverse primer sets for the STR locus of -179, consisting specifically of primer sets 1, 2 and 3.

In order to more intuitively reflect the technical solution of the present invention, the main points of the technical solutions described in the first to seventh above are summarized as follows:

Eighth, the present invention provides a kit for identifying complex kinship relationships, said kit comprising PCR primer combinations 1, 2, 3, 4, 5, 6 or 7 described in the description of the present invention.

In the primer combination, the concentrations of both forward and reverse primers are 0.1 μM.

Further, the kit also includes Index linker sequence and DNA polymerase;

Further preferably, the Index linker sequence includes IGT-I5Index and IGT-I7Index, and the concentration of the working solution is 10 μM.

The Index linker sequence is the linker sequence commonly used by the illumina sequencing platform, and is used to distinguish between samples.

Preferably, the kit also includes reagents for making genomic DNA into a library for sequencing.

Another aspect of the present invention also provides a method for identifying complex kinship, said method comprising the following steps:

(1) Human genomic DNA was extracted, and the concentration of quantitative genomic DNA was 1-20ng/μL;

(2) Multiplex PCR library construction:

A. After the first round of multiple PCR reaction and the first round of magnetic bead purification, the purified multiple PCR product is obtained;

B. Using the purified multiplex PCR product as a template, perform the second round of multiplex PCR reaction, insert the Index adapter sequence, and purify the second round of magnetic beads to obtain a multiplex PCR library;

C. Quantitative and quality detection of the obtained multiplex PCR library;

(3) Perform sequencing and data analysis on the multiplex PCR library obtained in step (2), and obtain the STR typing results and forensic parameters corresponding to each individual.

Further preferably, the concentration of genomic DNA is 10-20 ng/μL.

The STR typing system and kit are applicable to all samples containing DNA, including but not limited to blood, semen spots, hair, bones, skin, solid tissues, etc.

Compared with the prior art, the positive and beneficial effects of the present invention are:

(1) The STR typing system and its kit for the identification of complex kinship provided by the present invention can simultaneously detect 60, 58, 61, 118, 121, 119 or 179 linked autosomal STR genes at one time loci, in the process of establishing a multiplex PCR amplification system, as the number of detected STR loci increases, the mutual interference between each pair of primers will also increase. The present invention realizes the above-mentioned various numbers of Single-tube amplification of STR-linked autosomal STR loci, the STR typing system and its kits are well-balanced.

(2) In the prior art, independent inheritance of autosomal STR and sex chromosome-linked STR genetic markers cannot identify, for example, half-siblings and uncles (aunts), half-siblings and grandparents, or uncles and nephews and grandchildren The complex kinship relationship between different kinship relationships at the same level. However, the STR typing system and its kit provided by the present invention are aimed at the characteristics of linked autosomal STRs, and can be used to identify complex genetic relationships, and the identification results are accurate.

(3) The STR typing system and its kit provided by the present invention use two rounds of PCR reactions for library construction, which has many advantages such as short library construction cycle, high comparison rate, good capture efficiency, good repeatability, and easy operation, etc. The detection result has high sensitivity, good specificity, accurate typing result, and can effectively amplify 60, 58, 61, 118, 121, 119 or 179 linked autosomal STR loci at one time.

It should be clear that, on the basis that the present invention discloses all 179 STR loci and their corresponding amplified forward primers and reverse primers, those skilled in the art can, based on the content disclosed in the present invention, reasonably It is expected that any combination of the 179 STR sites described in the present invention has the number of combinations with genetic detection efficiency, or the technical solution composed of the combination of corresponding forward and reverse primers, can obtain the same or similar to the present invention. technical effect. The number of any genetic detection efficiency can be any integer between 3-179. Therefore, the technical solutions obtained through the methods described in this paragraph are all within the technical extension, protection scope and infringement scope of the present invention.

Description of drawings

FIG. 1 is a peak map of quality inspection of a library prepared using the primer combination disclosed in Example 1.

Fig. 2 is a quality inspection peak diagram of the library prepared by using the primer combination disclosed in Example 2.

Fig. 3 is a quality inspection peak map of the library prepared by using the primer combination disclosed in Example 3.

Fig. 4 is a quality inspection peak map of the library prepared by using the primer combination disclosed in Example 4.

Fig. 5 is a quality inspection peak map of the library prepared by using the primer combination disclosed in Example 5.

Fig. 6 is a peak map of quality inspection of the library prepared by using the primer combination disclosed in Example 6.

Fig. 7 is a quality inspection peak map of the library prepared by using the primer combination disclosed in Example 7.

Detailed ways

Specific embodiments of the present invention will be described in detail below. In order to avoid too many unnecessary details, well-known structures or functions will not be described in detail in the following embodiments. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1: A STR typing system and its kit for complex genetic relationship identification

1.1 Composition and characteristics of autosomal-linked STR loci:

(1) The STR typing system in this embodiment comprises the following 60 STR loci:

D1S2131、D1S3721、D1S2130、D1S1600、D1S1653、D1S1660、D1S3732、D2S1364、D2S2734、D2S1396、D2S428、D2S435、D2S1387、D2S1792、D2S1399、D2S2959、D3S2431、D3S4547、D4S1643、D4S2408、D4S3326、D4S2368、D5S2845、D5S1473、D5S813、 D5S1716、D5S1459、D5S1487、D5S1466、D5S2496、D5S2501、D6S1019、D6S2417、D6S2412、D6S1284、D7S1820、D7S3050、D7S821、D8S594、D8S1468、D8S2322、D8S569、D8S1475、D9S746、D9S319、D9S2149、D11S2010、D11S1392、D12S376、D12S1052、 D13S317, D13S790, D14S1432, D14S1428, D15S644, D15S1507, D16S752, D16S485, D20S481 and D20S1151;

It has the following measurable properties:

1) The repeating unit of the core region is tetranucleotide;

2) The heterozygosity of each STR is greater than 0.6;

3) Each group contains at least two STRs whose genetic distance is less than 3cM;

4) Each STR is named DXSXX.

(2) Primer combination 1 for simultaneously amplifying 60 STR loci in the present embodiment is:

Table 1: Forward primer set for primer set 1

Table 2: Reverse primer set for primer set 1

(3) In this embodiment, the working concentration of the forward primer is 0.1 μM; the working concentration of the reverse primer is 0.1 μM.

1.2 A kit for complex genetic relationship identification:

Including the above-mentioned PCR primer combination 1, Index linker sequence, and IGT-EM707 polymerase mixture.

The Index linker sequence includes IGT-I5Index and IGT-I7Index, and the concentration of the working solution is 10 μM.

The index linker sequence is: IGT-I7index I7-terminal linker sequence of IGT-I7, the working concentration is 10μM; one contains 96 links, and the linker sequence information is as follows

IGT-I5Index I5-terminal linker sequence of IGT-I5, the working concentration is 10μM; one contains 4, the linker sequence information is as follows

Note: The linker sequence is the common linker sequence of the illumina sequencing platform, which is used to distinguish between samples.

Example 2: A STR typing system and its kit for complex genetic relationship identification

2.1 Composition and characteristics of autosomal-linked STR loci:

(1) The STR typing system in this embodiment comprises the following 58 STR loci:

D1S3736、D1S1665、D1S2127、D1S2138、D1S1642、D1S518、D1S1604、D1S3729、D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、D2S2944、D2S1327、D3S2402、D3S1766、D4S2411、D4S3351、D4S243、D4S2426、D4S2373、D5S1490、D5S2856、 D5S2495、D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、D7S1799、D8S2324、D8S2330、D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、D11S1983、D11S2363、D11S1368、D12S393、D12S1063、D13S1807、D13S1492、D14S738、D14S301、 D14S583, D15S816, D15S1514, D18S872, D18S972, D18S548, D20S1145, D20S477;

It has the following measurable properties:

1) The repeating unit of the core region is tetranucleotide;

2) The heterozygosity of each STR is greater than 0.6;

3) Each group contains at least two STRs whose genetic distance is less than 3cM;

4) Each STR is named DXSXX.

(2) Primer combination 2 for simultaneously amplifying 58 STR loci in the present embodiment is:

Table 3: Forward primer set for primer set 2

Table 4: Reverse primer set for primer set 2

(3) In this embodiment, the working concentration of the forward primer is 0.1 μM; the working concentration of the reverse primer is 0.1 μM.

2.2 A kit for complex genetic relationship identification:

The only difference from Example 1 is that the PCR primer combination used is primer combination 2.

Example 3: A STR typing system and its kit for complex genetic relationship identification

3.1 Composition and characteristics of autosomal-linked STR loci:

(1) The STR typing system in this embodiment comprises the following 61 STR loci:

D1S532、D1S1611、D1S3733、D1S533、D1S1614、D2S2977、D2S1374、D2S1394、D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、D3S2388、D4S1626、D4S1653、D5S2858、D5S2796、D5S1463、D5S815、D5S2499、D5S2498、D6S1043、D6S1274、 D6S1056、D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、D8S2326、D8S1464、D8S2320、D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、D10S2469、D10S1238、D11S4464、D11S4958、D13S1818、D13S767、D14S615、D14S608、D14S597、 D14S302, D14S749, D16S767, D16S3393, D18S537, D18S875, D18S1367, D20S1152, D20S206, D20S607, D20S1146;

It has the following measurable properties:

1) The repeating unit of the core region is tetranucleotide;

2) The heterozygosity of each STR is greater than 0.6;

3) Each group contains at least two STRs whose genetic distance is less than 3cM;

4) Each STR is named DXSXX.

(2) Primer combination 3 for simultaneously amplifying 61 STR loci in the present embodiment is:

Table 5: Forward primer set for primer combination 3

Table 6: Reverse primer set for primer set 3

(3) In this embodiment, the working concentration of the forward primer is 0.1 μM; the working concentration of the reverse primer is 0.1 μM.

3.2 A kit for complex genetic relationship identification:

The only difference from Example 1 is that the PCR primer combination used is primer combination 3.

Embodiment 4: A kind of STR typing system and its kit for complex genetic relationship identification

4.1 Composition and characteristics of autosomal-linked STR loci:

(1) The STR typing system in this example contains 118 STR loci, specifically including the 60 STR loci described in Example 1 and the 58 STR loci described in Example 2.

It has the following measurable properties:

1) The repeating unit of the core region is tetranucleotide;

2) The heterozygosity of each STR is greater than 0.6;

3) Each group contains at least two STRs whose genetic distance is less than 3cM;

4) Each STR is named DXSXX.

(2) Primer combination 4 for simultaneously amplifying 118 STR loci in this example is a combination of primer combinations in Example 1 and Example 2.

(3) In this embodiment, the working concentration of the forward primer is 0.1 μM; the working concentration of the reverse primer is 0.1 μM.

4.2 A kit for complex genetic relationship identification:

The only difference from Example 1 is that the PCR primer combination used is primer combination 4.

Example 5: A STR typing system and its kit for complex genetic relationship identification

5.1 Composition and characteristics of autosomal-linked STR loci:

(1) The STR typing system in this example contains 121 STR loci, specifically including the 60 STR loci described in Example 1 and the 61 STR loci described in Example 3.

It has the following measurable properties:

1) The repeating unit of the core region is tetranucleotide;

2) The heterozygosity of each STR is greater than 0.6;

3) Each group contains at least two STRs whose genetic distance is less than 3cM;

4) Each STR is named DXSXX.

(2) Primer combination 5 for simultaneously amplifying 121 STR loci in this example is a combination of primer combinations in Example 1 and Example 3.

(3) In this embodiment, the working concentration of the forward primer is 0.1 μM; the working concentration of the reverse primer is 0.1 μM.

5.2 A kit for complex genetic relationship identification:

The only difference from Example 1 is that the PCR primer combination used is primer combination 5.

Example 6: A STR typing system and its kit for complex genetic relationship identification

6.1 Composition and characteristics of autosomal-linked STR loci:

(1) The STR typing system in this example contains 119 STR loci, specifically including the 58 STR loci described in Example 2 and the 61 STR loci described in Example 3.

It has the following measurable properties:

1) The repeating unit of the core region is tetranucleotide;

2) The heterozygosity of each STR is greater than 0.6;

3) Each group contains at least two STRs whose genetic distance is less than 3cM;

4) Each STR is named DXSXX.

(2) Primer combination 6 for simultaneously amplifying the 119 STR loci in this example is a combination of the primer combinations in Example 3 and Example 2.

(3) In this embodiment, the working concentration of the forward primer is 0.1 μM; the working concentration of the reverse primer is 0.1 μM.

6.2 A kit for complex genetic relationship identification:

The only difference from Example 1 is that the PCR primer combination used is primer combination 6.

Example 7: A STR Typing System for Identification of Complex Genetic Relationships

7.1 Composition and characteristics of autosomal-linked STR loci:

(1) The STR typing system in this example contains 179 STR loci, specifically including the 60 STR loci described in Example 1, the 58 STR loci described in Example 2, and the STR loci described in Example 3 61 STR loci.

It has the following measurable properties:

1) The repeating unit of the core region is tetranucleotide;

2) The heterozygosity of each STR is greater than 0.6;

3) Each group contains at least two STRs whose genetic distance is less than 3cM;

4) Each STR is named DXSXX.

(2) Primer combination 7 for simultaneously amplifying 179 STR loci in this example is a combination of primer combinations in Example 1, Example 3 and Example 2.

(3) In this embodiment, the working concentration of the forward primer is 0.1 μM; the working concentration of the reverse primer is 0.1 μM.

7.2 A kit for complex genetic relationship identification:

The only difference from Example 1 is that the PCR primer combination used is primer combination 7.

Experimental example

In the following experimental examples, the reagents used are legally available in the market, and the channels for obtaining them are as follows:

Tissue and blood DNA extraction kit: purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; article number: DP304-03;

QIAamp DNA Investigator Kit (50): purchased from Kaiser, Germany; item number: 5650;

Enhancer buffer NB (1N): PCR reaction enhancer named NB purchased from Aiji Taikang Company; product number: MT017035

IGT-EM707polymerase mixture: a DNA polymerase mixture named EM707 purchased from IGTEC; product number: MT017035;

YF Buffer B: The magnetic bead washing buffer named YF purchased from Aijitaikang; the article number is: MT017035;

The above reagents were custom-synthesized by Beijing iGeneTech ^TM , and the article number is: MT017035

Primer or Index sequence: Both are custom-synthesized by Beijing iGeneTech ^TM ;

1. Test samples: 108 unrelated individual samples of Han nationality in Beijing, including 48 blood samples and 60 FTA blood cards.

2. Detection object: The typing system and kit described in Examples 1-7 were used respectively, and the detection samples were detected according to the detection process.

3. Detection process:

(1) Whole-genome DNA was extracted from blood samples and FTA blood cards using tissues, blood DNA extraction kits and QIAamp DNA Investigator Kit (50) respectively. A nucleic acid quantifier was used for concentration determination, and the quantitative genomic DNA concentrations were 1, 5, 10, and 20 ng/μL.

(2) Multiplex PCR library construction:

The above-mentioned 1, 5, 10, and 20 ng/μL of genomic DNA were used for library construction, and the library numbers were F01, F02, F03, and F04, respectively.

A. The first round of multiple PCR reaction: Prepare the reaction mixture according to Table 7, 25 μL per tube, perform multiple PCR reactions according to the reaction conditions in Table 8, and obtain multiple PCR products; Refer to the combination of primers in Examples 1-7;

Table 7: The first round of multiplex PCR reaction system

Reagent Volume (μL) double distilled water 4 Enhancer buffer NB(1N) 7 Primer combination 8 Genomic DNA 1 IGT-EM707polymerase mixture 5

Table 8: Conditions for the first round of multiplex PCR reactions

The first round of magnetic bead purification:

Add 23 μL of AMPure XP magnetic beads equilibrated at room temperature to 25 μL of the multiplex PCR product, pipette and mix several times, incubate at room temperature for 5 minutes, place the PCR tube on the DynaMag-96Side magnetic stand for 3 minutes, and remove the supernatant completely , remove the PCR tube from the magnetic stand, add 40 μL of YF buffer B (magnetic bead washing buffer) and mix several times with a pipette, incubate at room temperature for 5 min, remove the supernatant, add 180 μL of 80% ethanol solution, and statically Leave it for 30s, remove the supernatant, add 180μL 80% ethanol solution, remove the supernatant completely after standing for 30s, and let it stand at room temperature for 3min to completely evaporate the residual ethanol, add 24μL Nuclease-free water (nuclease-free water) or 1 ×TE buffer (pH 8.0), pipette gently to resuspend the magnetic beads to avoid air bubbles, let it stand at room temperature for 2 minutes, put the PCR tube back on the magnetic stand and let it stand for 3 minutes, use a pipette to absorb the supernatant , transferred to a new PCR tube, and the supernatant in the tube was the purified multiplex PCR product.

B. The second round of multiple PCR reaction: use the purified multiple PCR product obtained in step A as a template to perform the second round of multiple PCR reaction, insert the Index linker sequence, and the second round of multiple PCR reaction system is as shown in Table 9, the reaction The conditions are as shown in Table 10;

Table 9: The second round of multiplex PCR reaction system

Reagent Volume (μL) Purified multiplex PCR products 18 IGT-I5Index (10μM) 1 IGT-I7Index (10μM) 1 IGT-EM707polymerase mixture 5

Table 10: The second round of multiplex PCR reaction conditions

The second round of magnetic bead purification:

The specific operation is the same as the first round of magnetic bead purification steps, and the supernatant transferred to a new PCR tube in the last step is the prepared multiplex PCR library.

C. Quantitative and quality detection of the obtained multiplex PCR library:

Take 1 μL of the multiplex PCR library obtained in step B, use a nucleic acid quantifier to measure the library concentration, and record the library concentration;

Take 1 μL of the multiplex PCR library obtained in step B, and use the automatic nucleic acid and protein analysis system to detect the length and purity of the library fragments. The target fragment distribution range of the normal library is 300bp-450bp, and the main peak is around 339bp.

(3) Perform next-generation sequencing on the multiplex PCR library obtained in step (2), use FASTQC software to perform quality control on the fastq files obtained by sequencing, and then use Trimmomatic software to filter the data, trim the sequences lower than Q30, and remove the sequencing fragments For sequences below 100bp, use the fastq file after quality control to type STR with STRaitRazor3.0, and finally get the STR typing file corresponding to each individual.

4. Test results:

(1) Using the typing system and kit disclosed in Examples 1-7, the typing and genetic relationship identification of 108 samples can be accurately realized.

(2), the quality inspection peak diagrams of the library prepared by using the primer combinations disclosed in Examples 1-7 are shown in Figures 1-7 respectively: it shows that the 7 STR typing systems and kits thereof provided by the present invention can be used in one-time , Simultaneously amplify the above 60, 58, 61, 118, 121, 119 or 179 STR loci, with good balance, good primer specificity and high sensitivity, and can effectively detect gDNA at a concentration as low as 1ng/μL.

(3) Effective typing information can be obtained for 108 samples by using the typing system and kit disclosed in Examples 1-7. In order to reasonably simplify the application documents, the typing information of 3 samples is listed here:

Analysis software: STRait Razor 3.0 software

Explanation of the table: the following blank box represents that the locus of the sample is homozygous, and the two typings represent heterozygotes; the number of sample reads after each typing represents the corresponding locus obtained by using the configuration file of STRait Razor 3.0 Typing coverage.

The typing results of the STR loci in Example 1 in the three samples are as follows:

The typing result information of the STR loci in the 3 samples in Example 2 is as follows:

The typing result information of the STR loci in the 3 samples in Example 3 is as follows:

The typing result of the above-mentioned 3 samples in embodiment 4 is equal to the result combination of embodiment 1 and embodiment 2; the typing result of the above-mentioned 3 samples in embodiment 5 is equal to the result combination of embodiment 1 and embodiment 3; Embodiment 6 is equal to the result combination of embodiment 2 and embodiment 3 to the typing result of above-mentioned 3 samples; Embodiment 7 is equal to embodiment 1, embodiment 2 and embodiment 3 to the typing result of above-mentioned 3 samples The results are combined; to avoid repetition, the table is not repeated here.

(4), typing efficiency:

The forensic parameters of 60, 58, 61, 118, 121, 119, and 179 STR sites contained in the typing system and kits disclosed in Examples 1-7 are as follows:

Analysis tools: STRAF 1.0.5: (STR Analysis for Forensics);

Analysis data source: typing result information of the above 108 irrelevant individual samples of Han nationality in Beijing;

Table explanation: Locus indicates the name of the STR locus; N indicates the number of alleles, a total of 108 Beijing Han unrelated individuals, so each STR genetic marker has 216 alleles; Nall indicates the total number of types of each locus, divided Type range is 5-19; GD (Genetic diversity, genetic polymorphism), its range is between 0.4911-0.9013; PIC (Polymorphism Information Content, polymorphism information content), its range is between 0.4368-0.8880; PM (match probability, matching probability), its range is between 0.0297-0.5111; PD (power of discrimination, personal identification probability), its range is between 0.4889-0.9703.

(5), library concentration:

The RFU (fluorescent signal intensity) values were slightly lower when the gDNA input amount was 1ng and 5ng, but they were all normal main peaks. When the gDNA input amount was 10ng and 20ng, not only the library mainly had normal main peaks but also the RFU value was better.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (10)

1. a kind of chain euchromosome STR classification system, which is characterized in that the chain euchromosome STR classification system packet Include the PCR primer combination 1 for expanding 60 chain euchromosome STR locus；

The chain euchromosome STR locus of described 60 be D1S2131, D1S3721, D1S2130, D1S1600, D1S1653, D1S1660、D1S3732、D2S1364、D2S2734、D2S1396、D2S428、D2S435、D2S1387、D2S1792、 D2S1399、D2S2959、D3S2431、D3S4547、D4S1643、D4S2408、D4S3326、D4S2368、D5S2845、 D5S1473、D5S813、D5S1716、D5S1459、D5S1487、D5S1466、D5S2496、D5S2501、D6S1019、 D6S2417、D6S2412、D6S1284、D7S1820、D7S3050、D7S821、D8S594、D8S1468、D8S2322、D8S569、 D8S1475、D9S746、D9S319、D9S2149、D11S2010、D11S1392、D12S376、D12S1052、D13S317、 D13S790,D14S1432,D14S1428,D15S644,D15S1507,D16S752,D16S485,D20S481,D20S1151；

The PCR primer combination 1 includes forward primer and reverse primer；

The forward primer is as follows:

The reverse primer is as follows:

2. chain euchromosome STR classification system according to claim 1, which is characterized in that the chain normal dyeing Body STR classification system further includes the PCR primer combination 2 for expanding 58 chain euchromosome STR locus；

The chain euchromosome STR locus of described 58 be D1S3736, D1S1665, D1S2127, D1S2138, D1S1642, D1S518、D1S1604、D1S3729、D1S3727、D2S1336、D2S1779、D2S1771、D2S437、D2S2970、 D2S2944、D2S1327、D3S2402、D3S1766、D4S2411、D4S3351、D4S243、D4S2426、D4S2373、 D5S1490、D5S2856、D5S2495、D5S2855、D5S1722、D5S1725、D6S1048、D6S1275、D7S796、 D7S1799、D8S2324、D8S2330、D8S1144、D9S745、D9S301、D9S1124、D10S1246、D10S2485、 D11S1983、D11S2363、D11S1368、D12S393、D12S1063、D13S1807、D13S1492、D14S738、 D14S301,D14S583,D15S816,D15S1514,D18S872,D18S972,D18S548,D20S1145,D20S477；

The PCR primer combination 2 includes forward primer and reverse primer；

The forward primer is as follows:

The reverse primer is as follows:

3. chain euchromosome STR classification system according to claim 2, which is characterized in that the chain normal dyeing Body STR classification system further includes the PCR primer combination 3 for expanding 61 chain euchromosome STR locus；

The chain euchromosome STR locus of described 61 be D1S532, D1S1611, D1S3733, D1S533, D1S1614, D2S2977、D2S1374、D2S1394、D2S2966、D2S2969、D2S1371、D2S434、D2S1338、D3S4016、 D3S2388、D4S1626、D4S1653、D5S2858、D5S2796、D5S1463、D5S815、D5S2499、D5S2498、 D6S1043、D6S1274、D6S1056、D6S1013、D6S1054、D7S820、D7S2205、D7S3071、D7S2845、 D8S2326、D8S1464、D8S2320、D8S1470、D8S588、D8S1471、D9S2026、D9S747、D9S2128、 D10S2469、D10S1238、D11S4464、D11S4958、D13S1818、D13S767、D14S615、D14S608、D14S597、 D14S302、D14S749、D16S767、D16S3393、D18S537、D18S875、D18S1367、D20S1152、D20S206、 D20S607,D20S1146；

The PCR primer combination 3 includes forward primer and reverse primer；

The forward primer is as follows:

The reverse primer is as follows:

4. any more than 30 str locus seats and its forward direction in the str locus seat as described in any one of claim 1-3 The STR classification system constituted with reverse primer.

5. a kind of kit for complicated Relationship iden- tification, which is characterized in that the kit includes claim 1 institute State for expand the PCR primer combinations 1 of 60 chain euchromosome STR locus, in the PCR primer forward primer with The working concentration of reverse primer is 0.1 μM.

6. kit according to claim 5, which is characterized in that the kit further includes as claimed in claim 2 For expanding the PCR primer combination 2 of 58 chain euchromosome STR locus, middle forward primer and anti-in the PCR primer It is 0.1 μM to the working concentration of primer.

7. kit according to claim 6, which is characterized in that the kit further includes as claimed in claim 3 For expanding the PCR primer combination 3 of 61 chain euchromosome STR locus, middle forward primer and anti-in the PCR primer It is 0.1 μM to the working concentration of primer.

8. according to kit described in claim 5-7 any one, which is characterized in that the kit further includes Index Joint sequence and archaeal dna polymerase.

9. kit according to claim 8, which is characterized in that the Index joint sequence includes IGT-I5 Index and IGT-I7 Index, working concentration are 10 μM.

10. kit according to claim 8, which is characterized in that the kit further includes that genomic DNA is made For the reagent in the library of sequencing.