CN112176068B - A composite amplification system based on 29 Y-STR loci and primer combination used therefor - Google Patents

Abstract

The invention discloses a multiplex amplification system based on 29Y-STR loci and a primer combination used by the same. The primer combination provided by the invention consists of 52 DNA molecules shown by a sequence 1 to a sequence 52 in a sequence table. The primer combination is adopted to construct a composite amplification system, then STR typing of male individuals is carried out, and the detected polymorphism is high. Meanwhile, the 5 rapid mutation Y-STR loci and the 7 Chinese population Y-STR loci are medium mutation rate Y-STR loci with higher resolution in the Chinese population, so that the composite amplification system not only has higher resolution and better compatibility, but also has better pedigree representativeness, is more suitable for the Chinese population, and can greatly improve the resolution and the recognition capability of the system. The invention has important application value.

Description

A composite amplification system based on 29 Y-STR loci and primer combination used therefor

technical field

The invention belongs to the technical field of forensic medicine, in particular to a compound amplification system based on 29 Y-STR loci and a primer combination used therefor, in particular to a Y-STR based on 17 general Y-STR loci and 5 rapid mutation Y-STRs A composite amplification system of the locus and seven Chinese population Y-STR loci and the primer combinations used.

Background technique

The Y chromosome is male-specific, and all male individuals of the same paternal line have the same or similar Y-STR haplotype. Based on its paternal genetic characteristics, the pioneered Y-STR family screening method is a new attempt to apply Y-STR technology in criminal investigation and solving cases, and also provides a new working idea for the current public security organs to detect cases in a timely manner. The Y-STR family investigation method means that after the investigators carry out effective Y-STR typing on the collected biological materials left behind by the male perpetrator, they will search within a certain geographical scope or in the Y-STR database to search for the same or similar physical evidence as the on-site physical evidence. It is an investigation method in which male individuals with similar Y-STR typing can find out the family of the perpetrator, and then use autosomal detection and other technical means to accurately find the perpetrator in the family. At present, Y-STR pedigree investigation has become an important means of case investigation, especially in some cases in remote areas where families live together and people flow less, Y-STR investigation provides a very important direction for the detection of cases sexual cues. The Y-STR detection reagent is used to detect male genetic characteristics, which plays a very important role in general case inspection and Y family investigation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to perform STR typing in male individuals.

The present invention first protects primer combinations, which may include primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14 , primer 15, primer 16, primer 17, primer 18, primer 19, primer 20, primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer 28, primer 29, primer 30, primer 31, primer 32, primer 33, primer 34, primer 35, primer 36, primer 37, primer 38, primer 39, primer 40, primer 41, primer 42, primer 43, primer 44, primer 45, primer 46, primer 47, Primer 48, Primer 49, Primer 50, Primer 51 and Primer 52;

Primer 1 is the single-stranded DNA molecule shown in Sequence 1 in the sequence listing;

Primer 2 is the single-stranded DNA molecule shown in sequence 2 in the sequence listing;

Primer 3 is the single-stranded DNA molecule shown in sequence 3 in the sequence listing;

Primer 4 is the single-stranded DNA molecule shown in sequence 4 in the sequence listing;

Primer 5 is the single-stranded DNA molecule shown in sequence 5 in the sequence listing;

Primer 6 is the single-stranded DNA molecule shown in sequence 6 in the sequence listing;

Primer 7 is the single-stranded DNA molecule shown in sequence 7 in the sequence listing;

Primer 8 is the single-stranded DNA molecule shown in sequence 8 in the sequence listing;

Primer 9 is the single-stranded DNA molecule shown in sequence 9 in the sequence listing;

Primer 10 is the single-stranded DNA molecule shown in sequence 10 in the sequence listing;

Primer 11 is the single-stranded DNA molecule shown in sequence 11 in the sequence listing;

Primer 12 is the single-stranded DNA molecule shown in sequence 12 in the sequence listing;

Primer 13 is the single-stranded DNA molecule shown in sequence 13 in the sequence listing;

Primer 14 is the single-stranded DNA molecule shown in sequence 14 in the sequence listing;

Primer 15 is the single-stranded DNA molecule shown in sequence 15 in the sequence listing;

Primer 16 is the single-stranded DNA molecule shown in sequence 16 in the sequence listing;

Primer 17 is the single-stranded DNA molecule shown in sequence 17 in the sequence listing;

Primer 18 is the single-stranded DNA molecule shown in sequence 18 in the sequence listing;

Primer 19 is the single-stranded DNA molecule shown in sequence 19 in the sequence listing;

Primer 20 is the single-stranded DNA molecule shown in sequence 20 in the sequence listing;

Primer 21 is the single-stranded DNA molecule shown in sequence 21 in the sequence listing;

Primer 22 is the single-stranded DNA molecule shown in sequence 22 in the sequence listing;

Primer 23 is the single-stranded DNA molecule shown in sequence 23 in the sequence listing;

Primer 24 is the single-stranded DNA molecule shown in sequence 24 in the sequence listing;

Primer 25 is the single-stranded DNA molecule shown in sequence 25 in the sequence listing;

Primer 26 is the single-stranded DNA molecule shown in sequence 26 in the sequence listing;

Primer 27 is the single-stranded DNA molecule shown in sequence 27 in the sequence listing;

Primer 28 is the single-stranded DNA molecule shown in sequence 28 in the sequence listing;

Primer 29 is the single-stranded DNA molecule shown in sequence 29 in the sequence listing;

The primer 30 is the single-stranded DNA molecule shown in the sequence 30 in the sequence listing;

Primer 31 is the single-stranded DNA molecule shown in sequence 31 in the sequence listing;

Primer 32 is the single-stranded DNA molecule shown in sequence 32 in the sequence listing;

Primer 33 is the single-stranded DNA molecule shown in sequence 33 in the sequence listing;

Primer 34 is the single-stranded DNA molecule shown in sequence 34 in the sequence listing;

Primer 35 is the single-stranded DNA molecule shown in sequence 35 in the sequence listing;

Primer 36 is the single-stranded DNA molecule shown in sequence 36 in the sequence listing;

Primer 37 is the single-stranded DNA molecule shown in sequence 37 in the sequence listing;

Primer 38 is the single-stranded DNA molecule shown in sequence 38 in the sequence listing;

Primer 39 is the single-stranded DNA molecule shown in sequence 39 in the sequence listing;

The primer 40 is the single-stranded DNA molecule shown in the sequence 40 in the sequence listing;

Primer 41 is the single-stranded DNA molecule shown in sequence 41 in the sequence listing;

Primer 42 is the single-stranded DNA molecule shown in sequence 42 in the sequence listing;

Primer 43 is the single-stranded DNA molecule shown in sequence 43 in the sequence listing;

Primer 44 is the single-stranded DNA molecule shown in sequence 44 in the sequence listing;

Primer 45 is the single-stranded DNA molecule shown in sequence 45 in the sequence listing;

Primer 46 is the single-stranded DNA molecule shown in sequence 46 in the sequence listing;

Primer 47 is the single-stranded DNA molecule shown in sequence 47 in the sequence listing;

Primer 48 is the single-stranded DNA molecule shown in sequence 48 in the sequence listing;

Primer 49 is the single-stranded DNA molecule shown in sequence 49 in the sequence listing;

The primer 50 is the single-stranded DNA molecule shown in the sequence 50 in the sequence listing;

Primer 51 is the single-stranded DNA molecule shown in sequence 51 in the sequence listing;

Primer 52 is a single-stranded DNA molecule represented by SEQ ID NO: 52 in the Sequence Listing.

The primer combination can be specifically composed of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, primer 15 , primer 16, primer 17, primer 18, primer 19, primer 20, primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer 28, primer 29, primer 30, primer 31, primer 32, primer 33, primer 34, primer 35, primer 36, primer 37, primer 38, primer 39, primer 40, primer 41, primer 42, primer 43, primer 44, primer 45, primer 46, primer 47, primer 48, It consists of primer 49, primer 50, primer 51 and primer 52.

In any of the primer combinations described above, primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, primer 15, primer 16, primer 17, primer 18, primer 19, primer 20, primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer 28, primer 29, primer 30, Primer 31, Primer 32, Primer 33, Primer 34, Primer 35, Primer 36, Primer 37, Primer 38, Primer 39, Primer 40, Primer 41, Primer 42, Primer 43, Primer 44, Primer 45, Primer 46, Primer 47 , primer 48, primer 49, primer 50, primer 51 and primer 52 in a molar ratio of 35:35:75:75:75:75:100:100:100:100:125:125:70:70:55: 55:40:40:90:90:100:100:100:100:125:125:50:50:65:65:55:55:90:90:100:100:125:125:45:45: 65:65:50:50:90:90:90:90:100:100:150:150.

In any of the primer combinations described above, primer 1, primer 3, primer 5, primer 7, primer 9, primer 11, primer 13, primer 15, primer 17, primer 19, primer 21, primer 23, primer 25, primer 27. Primer 29, primer 31, primer 33, primer 35, primer 37, primer 39, primer 41, primer 43, primer 45, primer 47, primer 49 and primer 51 were all fluorescently labeled.

In any one of the primer combinations described above, primer 1, primer 3, primer 5, primer 7, primer 9 and primer 11 are labeled with FAM. Primer 13, Primer 15, Primer 17, Primer 19, Primer 21, Primer 23 and Primer 25 were labeled with HEX. Primer 27, primer 29, primer 31, primer 33, primer 35 and primer 37 were labeled with TAMRA. Primer 39, primer 41, primer 43, primer 45, primer 47, primer 49 and primer 51 were labeled with ROX. The 5' ends of primer 1, primer 3, primer 5, primer 7, primer 9 and primer 11 were labeled with FAM. The 5' ends of primer 13, primer 15, primer 17, primer 19, primer 21, primer 23 and primer 25 were labeled with HEX. The 5' ends of primer 27, primer 29, primer 31, primer 33, primer 35 and primer 37 were labeled with TAMRA. The 5' ends of primer 39, primer 41, primer 43, primer 45, primer 47, primer 49 and primer 51 were labeled with ROX.

The present invention also protects a composite amplification system based on the Y-STR locus, which can include any one of the above-mentioned primer combinations;

The Y-STR loci can include DYS460, DYS389I/II, DYS390, DYS533, DYS392, DYS518, DYS508, DYS437, DYS458, DYS385ab, GATA-H4, DYS576, DYS641, DYS456, DYS391, DYS447, DYS438, DYS448, DYF3877 , DYS393, DYS635, DYS439, DYS19, DYS444, DDYS449 and DYS481.

DYS449, DYS518, DYS576 and DYF387S1 are all rapidly mutating Y-STR loci.

The composite amplification system may specifically be composed of any one of the above-mentioned primer combinations.

In any of the above composite amplification systems, the concentration of primer 1 and primer 2 in the composite amplification system may be 35 mM. The concentration of primer 3, primer 4, primer 5 and primer 6 in the composite amplification system may be 75 mM. The concentration of primer 7, primer 8, primer 9, primer 10, primer 21, primer 22, primer 23, primer 24, primer 35, primer 36, primer 49 and primer 50 in the composite amplification system may be 100 mM. The concentration of primer 11, primer 12, primer 25, primer 26, primer 37 and primer 38 in the composite amplification system may be 125 mM. The concentration of primer 13 and primer 14 in the composite amplification system may be 70 mM. The concentration of primer 15, primer 16, primer 31 and primer 32 in the composite amplification system may be 55 mM. The concentration of primer 17 and primer 18 in the composite amplification system may be 40 mM. The concentration of primer 19, primer 20, primer 33, primer 34, primer 45, primer 46, primer 47 and primer 48 in the composite amplification system may be 90 mM. The concentration of primer 27, primer 28, primer 43 and primer 44 in the complex amplification system may be 50 mM. The concentration of primer 29, primer 30, primer 41 and primer 42 in the composite amplification system may be 65 mM. The concentration of primer 39 and primer 40 in the composite amplification system may be 45 mM. The concentration of primer 51 and primer 52 in the composite amplification system may be 150 mM.

In the above composite amplification system, the composite amplification system may further include reagents required for PCR amplification reaction; the "reagents required for PCR amplification reaction" do not include primers required for PCR amplification reaction.

The "reagents required for PCR amplification reaction" may include at least one of DNA polymerase, dNTP, Mg ² +, BSA, KCl, and Tris.

The concentration of the DNA polymerase in the composite amplification system may be 0.1 U/μL. The concentration of the dNTP in the composite amplification system may be 200 μM (ie, the concentration of dATP, dTTP, dCTP and dGTP is 200 μM). ^The concentration of the Mg 2+ in the composite amplification system may be 1.5 mM. The concentration of the BSA in the composite amplification system may be 0.8 mg/mL. The concentration of the KCl in the composite amplification system may be 50 mM. The concentration of the Tris in the composite amplification system may be 10 mM.

The composite amplification system may specifically be composed of any of the above-mentioned primer combinations and reagents required for PCR amplification reaction.

A kit containing any of the above-mentioned primer combinations also belongs to the protection scope of the present invention. The kit is used for STR typing of male individuals.

The preparation method of any of the above-mentioned composite amplification systems or the kit containing any of the above-mentioned primer combinations also belongs to the protection scope of the present invention.

The preparation method of any of the above-mentioned composite amplification systems or the kit containing any of the above-mentioned primer combinations may include the step of individually packaging each primer in any of the above-mentioned primer combinations.

The following X1) or X2) also belong to the protection scope of the present invention.

X1) Application of any one of the above-mentioned primer combinations or any of the above-mentioned composite amplification systems in preparing a kit for STR typing of male individuals.

X2) Application of any one of the above-mentioned primer combinations or any of the above-mentioned composite amplification systems in STR typing of male individuals.

Any of the above STR typing may specifically be Y-STR typing.

Any of the male individuals described above may be Chinese male individuals.

The composite amplification system provided by the present invention is used to perform STR typing on sample one (9948 human genomic DNA) or sample two (blood collection card of a human (known as a male)). The results showed that both samples 1 and 2 obtained complete STR typing, and the peaks were sharp and clear, with good balance, no pull-up peaks, stutter bands, and no non-specific amplification products. The results of 9948 human genomic DNA and Ladder also showed that the compound amplification system showed that the 29 Y-STR loci shown in Table 1 (including 20 core loci, 8 preferred loci and The genotyping result of 1 candidate locus) is correct, which can fully meet the requirements of forensic Y-STR test. It can be seen that the composite amplification system provided by the present invention can be used to amplify the 29 Y-STR loci shown in Table 1 (5 of which are rapidly mutated Y-STR loci and 7 are Chinese population Y-STR genes locus), the composite amplification system can obtain up to 29 different allele fragments in one amplification, and the amplification efficiency is very high. At the same time, the 5 rapidly mutating Y-STR loci and the 7 Chinese population Y-STR loci are all Y-STR loci with high resolution in the Chinese population with a moderate mutation rate, so the composite amplification system provided by the present invention It not only has higher resolution and better compatibility, but also has better family representativeness, which is more suitable for Chinese people, which can greatly improve the resolution and recognition ability of the system. In addition, the composite amplification system is suitable for 3130, 3500, 3730 and other types of sequencers, and all loci are less than 470bp, which has good adaptability to degraded samples. The invention has important application value.

Description of drawings

Figure 1 shows the DNA detection map of sample one.

Figure 2 shows the DNA detection map of sample two.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

9948 human genomic DNA is a product of Promaga.

Example 1. Preparation of composite amplification system based on 29 Y-STR loci

1. Screening of Y-STR loci

The inventors of the present invention obtained a large number of candidate Y-STR loci by consulting the literature; then applied the research results of the Human Genome Project, and used the reported various loci on the UCSC website (URL: http://genome.ucsc.edu/). The locus primer information was searched to obtain the corresponding sequence; the sequence was searched in GenBank by BLAST (website: https://blast.ncbi.nlm.nih.gov/Blast.cgi), and then the existing The core and flanking region sequence information, allele length and distribution of the Y-STR locus were sorted out and systematically studied, focusing on the Y-STR locus with a core sequence of 4-6 base repeats. Considering technical indicators such as the copy number, mutation rate, repeat unit characteristics, sequence structure complexity of each locus, and compatibility with other commercial kits, 29 Y-STR loci were finally screened, including 17 Universal Y-STR loci, 5 rapidly mutating Y-STR loci and 7 Chinese population Y-STR loci.

The 17 universal Y-STR loci are: DYS19, DYS385ab, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA-H4, DYS438, DYS439.

Five rapidly mutating Y-STR loci: DYS449, DYS518, DYS576, DYF387S1.

Seven Chinese population Y-STR loci: DYS460, DYS447, DYS444, DYS508, DYS533, DYS643, DYS481.

It should be noted that there is no significant difference in the lengths of amplified fragments of DYS385ab and DYF387S1, which are traditionally distinguished by a/b, that is, DYS385ab is divided into DYS385a and DYS385b, DYF387S1 is divided into DYF387S1a and DYF387S1b; the lengths of amplified fragments of DYS389I/II are The obvious difference is usually distinguished by I/II, that is, DYS389I/II is divided into DYS389I and DYS389II.

2. Preparation of a composite amplification system based on 29 Y-STR loci

1. Preliminarily screen out suitable fluorescent dyes according to the emission wavelength range of each fluorescent dye and the intensity of fluorescent signals, and then test the stability of fluorescent dyes through a large number of experiments, and finally obtain five-color fluorescent dyes that do not interfere with each other and have stable quality. Five-color fluorescence detection.

2. According to the allele composition, range and amplified fragment length of each locus (all less than 470bp), reasonably select fluorescent markers of different colors to distinguish each locus located in the overlapping region of the fragments, and then prepare the 29 Y-STRs based on 29 Y-STRs. A complex amplification system for loci. Specific steps are as follows:

(1) Artificially synthesize primers for amplifying each locus, and then perform PCR amplification to obtain specific primers for each locus.

(2) Combine the amplification conditions of a single locus, select an appropriate amplification procedure, firstly compound amplification with primers labeled with the same color fluorescence, and then compound amplification with primers labeled with different colors of fluorescence after confirmation. During compound amplification, observe whether there are specific products and non-specific products at each locus. If there are non-specific spurious peaks between primers of different loci, it is necessary to exclude them one by one, find these loci, redesign and synthesize primers. In addition, primer dimers will be formed between primers of different loci, which reduces the amplification efficiency of primers, and it is necessary to redesign and synthesize primers.

(3) Repeatedly test and modify the composite amplification system, and use the composite amplification system to amplify the male genomic DNA to observe whether there is overlap between the loci, because the amplification product is designed according to the theoretical size, and the actual process Due to the inconsistent sequence structure of the amplified products, the migration rate is too fast or too slow, resulting in a discrepancy between the theoretical size and the actual size, often causing overlapping or too close distances between adjacent loci, affecting subsequent analysis. Once such a phenomenon occurs, it is necessary to redesign the primers of the locus, and then go through steps (1) and (2) to finally obtain a complex amplification system that meets the conditions.

(4) Leveling of the compound amplification system. Specifically: in the initial composite amplification, the concentration of each primer in the reaction system is 100 mM; the concentration of each primer pair is adjusted according to the reaction result.

(5) Optimize the parameters of the composite amplification reaction. Use 9948 human genomic DNA to adjust the key components and links in the reaction system, such as the optimal reaction system, number of amplification cycles, annealing temperature, extension time, amount of hot-start DNA polymerase, amount of primers, buffer concentration, etc. .

After the above steps, a composite amplification system based on 29 Y-STR loci was obtained. The composite amplification system is composed of DNA polymerase, dNTP, Mg ²⁺ , BSA, KCl, Tris and primer mixture; the primer mixture is composed of 52 primers; the names, nucleotide sequences, alleles of the 52 primers and amplified loci, etc., see columns 1-5 in Table 1. In this composite amplification system, the concentration of DNA polymerase was 0.1 U/μL, the concentration of dNTP was 200 μM (that is, the concentrations of dATP, dTTP, dCTP and dGTP were all 200 μM), the concentration of Mg ²⁺ was 1.5 mM, and the concentration of BSA was 1.5 mM. The concentration is 0.8 mg/mL, the concentration of KCl is 50 mM, the concentration of Tris is 10 mM, and the concentration of 52 primers is shown in column 6 in Table 1.

Table 1

Note: FAM means FAM labeling at the 5' end; HEX means HEX labeling at the 5' end; TAMRA means TAMRA labeling at the 5' end; ROX means ROX labeling at the 5' end.

3. Prepare the above-mentioned composite amplification system based on 29 Y-STR loci.

Example 2. Application of compound amplification system based on 29 Y-STR loci

Typer500 is the product of the Ministry of Public Security's Physical Evidence Identification Center. Deionized formamide is a product of ABI Corporation, catalog number 4311320. ABI3130xl Genetic Analyzer is a product of ABI Corporation. The blood collection card of a person (known as a male) is provided by the construction of the daily DNA database of the Physical Evidence Identification Center of the Ministry of Public Security.

The sample to be tested is sample 1 or sample 2.

Sample 1: 1ng of 9948 human genomic DNA.

Sample 2: Take a blood collection card from a person (known as a male), and punch a circular piece with a 0.5mm hand-held punch.

1. Take 10 μL of the composite amplification system prepared in step 2, step 2 of Example 1, add the sample to be tested for PCR amplification, and obtain a PCR amplification product.

The reaction program was as follows: pre-denaturation at 95 °C for 11 min; denaturation at 94 °C for 30 s, annealing at 59 °C for 2 min, extension at 72 °C for 1 min, 28 cycles; extension at 60 °C for 60 min; storage at 4 °C.

2. After completing step 1, mix 10 μL of the sample mixture and 1 μL of the PCR amplification product evenly to obtain a reaction solution.

The loading mixture was composed of 10 μL Typer500 internal standard and 1000 μL deionized formamide.

3. After completing step 2, take the reaction solution, denature it at 95°C for 5 minutes, quickly transfer it to -20°C for 5 minutes, and then perform capillary electrophoresis detection with ABI3130xl genetic analyzer to obtain a DNA detection map. The electrophoresis conditions were as follows: the injection voltage was 1.2 kV, and the injection time was 30 s.

The test results of sample one are shown in Figure 1.

The test results of sample two are shown in Figure 2.

The results showed that both samples 1 and 2 obtained complete STR typing, and the peaks were sharp and clear, with good balance, no pull-up peaks, stutter bands, and no non-specific amplification products. The results of 9948 human genomic DNA also show that the compound amplification system prepared in Example 1 has correct typing results for the 29 Y-STR loci shown in Table 1, and can fully meet the requirements of forensic Y-STR testing.

The composite amplification system provided by the present invention can be used to amplify the 29 Y-STR loci shown in Table 1 (5 of which are rapidly mutated Y-STR loci, and 7 are Chinese population Y-STR loci). The composite amplification system can obtain up to 29 different allele fragments in one amplification, and the amplification efficiency is very high. At the same time, the 5 rapidly mutating Y-STR loci and the 7 Chinese population Y-STR loci are all Y-STR loci with high resolution in the Chinese population with a moderate mutation rate, so the composite amplification system provided by the present invention It not only has higher resolution and better compatibility, but also has better family representativeness, which is more suitable for Chinese people, which can greatly improve the resolution and recognition ability of the system.

<110> Ministry of Public Security Material Evidence Identification Center

<120> A composite amplification system based on 29 Y-STR loci and primer combinations used therefor

<160> 52

<170> PatentIn version 3.5

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<213> Artificial sequence

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atggaatgct ctcttggctt c 21

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence

<400> 42

gcccaaatat ccatcaatca 20

<210> 43

<211> 24

<212> DNA

<213> Artificial sequence

<400> 43

tcgagttgtt atggttttag gtct 24

<210> 44

<211> 22

<212> DNA

<213> Artificial sequence

<400> 44

gcttggaatt cttttaccca tc 22

<210> 45

<211> 22

<212> DNA

<213> Artificial sequence

<400> 45

gtcaatctct gcacctggaa at 22

<210> 46

<211> 26

<212> DNA

<213> Artificial sequence

<400> 46

atgactactg agtttctgtt atgactg 26

<210> 47

<211> 22

<212> DNA

<213> Artificial sequence

<400> 47

atgaaaggtg tgaaccattt gg 22

<210> 48

<211> 24

<212> DNA

<213> Artificial sequence

<400> 48

tccaaaggca gaaggaaatc tata 24

<210> 49

<211> 21

<212> DNA

<213> Artificial sequence

<400> 49

caggagacag cctgttctat g 21

<210> 50

<211> 23

<212> DNA

<213> Artificial sequence

<400> 50

ctggaagtgg agtttgctgt aag 23

<210> 51

<211> 20

<212> DNA

<213> Artificial sequence

<400> 51

aggaatgtgg ctaacgctgt 20

<210> 52

<211> 25

<212> DNA

<213> Artificial sequence

<400> 52

gcagagtgaa atacattttc ccctg 25

Claims (10)

1. A primer combination consisting of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, primer 15, primer 16, primer 17, primer 18, primer 19, primer 20, primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer 28, primer 29, primer 30, primer 31, primer 32, primer 33, primer 34, primer 35, primer 36, primer 37, primer 38, primer 39, primer 40, primer 41, primer 42, primer 43, primer 44, primer 45, primer 46, primer 47, primer 48, primer 49, primer 50, primer 51 and primer 52;

the primer 1 is a single-stranded DNA molecule shown as a sequence 1 in a sequence table;

the primer 2 is a single-stranded DNA molecule shown as a sequence 2 in a sequence table;

the primer 3 is a single-stranded DNA molecule shown as a sequence 3 in a sequence table;

the primer 4 is a single-stranded DNA molecule shown as a sequence 4 in a sequence table;

the primer 5 is a single-stranded DNA molecule shown as a sequence 5 in the sequence table;

the primer 6 is a single-stranded DNA molecule shown as a sequence 6 in a sequence table;

the primer 7 is a single-stranded DNA molecule shown as a sequence 7 in a sequence table;

the primer 8 is a single-stranded DNA molecule shown as a sequence 8 in a sequence table;

the primer 9 is a single-stranded DNA molecule shown as a sequence 9 in a sequence table;

the primer 10 is a single-stranded DNA molecule shown as a sequence 10 in a sequence table;

the primer 11 is a single-stranded DNA molecule shown as a sequence 11 in a sequence table;

the primer 12 is a single-stranded DNA molecule shown as a sequence 12 in a sequence table;

the primer 13 is a single-stranded DNA molecule shown as a sequence 13 in a sequence table;

the primer 14 is a single-stranded DNA molecule shown as a sequence 14 in a sequence table;

the primer 15 is a single-stranded DNA molecule shown as a sequence 15 in a sequence table;

the primer 16 is a single-stranded DNA molecule shown as a sequence 16 in a sequence table;

the primer 17 is a single-stranded DNA molecule shown as a sequence 17 in a sequence table;

the primer 18 is a single-stranded DNA molecule shown as a sequence 18 in a sequence table;

the primer 19 is a single-stranded DNA molecule shown as a sequence 19 in a sequence table;

the primer 20 is a single-stranded DNA molecule shown as a sequence 20 in a sequence table;

the primer 21 is a single-stranded DNA molecule shown as a sequence 21 in a sequence table;

the primer 22 is a single-stranded DNA molecule shown as a sequence 22 in a sequence table;

the primer 23 is a single-stranded DNA molecule shown as a sequence 23 in a sequence table;

the primer 24 is a single-stranded DNA molecule shown as a sequence 24 in a sequence table;

the primer 25 is a single-stranded DNA molecule shown as a sequence 25 in a sequence table;

the primer 26 is a single-stranded DNA molecule shown as a sequence 26 in a sequence table;

the primer 27 is a single-stranded DNA molecule shown as a sequence 27 in a sequence table;

the primer 28 is a single-stranded DNA molecule shown as a sequence 28 in a sequence table;

the primer 29 is a single-stranded DNA molecule shown as a sequence 29 in a sequence table;

the primer 30 is a single-stranded DNA molecule shown as a sequence 30 in a sequence table;

the primer 31 is a single-stranded DNA molecule shown as a sequence 31 in a sequence table;

the primer 32 is a single-stranded DNA molecule shown as a sequence 32 in a sequence table;

the primer 33 is a single-stranded DNA molecule shown as a sequence 33 in a sequence table;

the primer 34 is a single-stranded DNA molecule shown as a sequence 34 in a sequence table;

the primer 35 is a single-stranded DNA molecule shown as a sequence 35 in a sequence table;

the primer 36 is a single-stranded DNA molecule shown as a sequence 36 in a sequence table;

the primer 37 is a single-stranded DNA molecule shown as a sequence 37 in a sequence table;

the primer 38 is a single-stranded DNA molecule shown as a sequence 38 in a sequence table;

the primer 39 is a single-stranded DNA molecule shown as a sequence 39 in a sequence table;

the primer 40 is a single-stranded DNA molecule shown as a sequence 40 in a sequence table;

the primer 41 is a single-stranded DNA molecule shown as a sequence 41 in a sequence table;

the primer 42 is a single-stranded DNA molecule shown as a sequence 42 in a sequence table;

the primer 43 is a single-stranded DNA molecule shown as a sequence 43 in a sequence table;

the primer 44 is a single-stranded DNA molecule shown as a sequence 44 in a sequence table;

the primer 45 is a single-stranded DNA molecule shown as a sequence 45 in a sequence table;

the primer 46 is a single-stranded DNA molecule shown as a sequence 46 in a sequence table;

the primer 47 is a single-stranded DNA molecule shown as a sequence 47 in a sequence table;

the primer 48 is a single-stranded DNA molecule shown as a sequence 48 in a sequence table;

the primer 49 is a single-stranded DNA molecule shown as a sequence 49 in a sequence table;

the primer 50 is a single-stranded DNA molecule shown as a sequence 50 in a sequence table;

the primer 51 is a single-stranded DNA molecule shown as a sequence 51 in a sequence table;

the primer 52 is a single-stranded DNA molecule shown as a sequence 52 in a sequence table.

2. The primer combination of claim 1, wherein: primer 1, primer 2, primer 3, primer 4, primer 5, primer 6, primer 7, primer 8, primer 9, primer 10, primer 11, primer 12, primer 13, primer 14, primer 15, primer 16, primer 17, primer 18, primer 19, primer 20, primer 21, primer 22, primer 23, primer 24, primer 25, primer 26, primer 27, primer 28, primer 29, primer 30, primer 31, primer 32, primer 33, primer 34, primer 35, primer 36, primer 37, primer 38, primer 39, primer 40, primer 41, primer 42, primer 43, primer 44, primer 45, primer 46, primer 47, primer 48, primer 49, primer 50, primer 51 and primer 52 are present in a molar ratio of 35: 35: 75: 75: 75: 75: 100: 100: 100: 100: 125: 125: 70: 70: 55: 55: 40: 40: 90: 90: 100: 100: 100: 100: 125: 125: 50: 50: 65: 65: 55: 55: 90: 90: 100: 100: 125: 125: 45: 45: 65: 65: 50: 50: 90: 90: 90: 90: 100: 100: 150: 150.

3. the primer combination of claim 1 or 2, wherein: primer 1, primer 3, primer 5, primer 7, primer 9, primer 11, primer 13, primer 15, primer 17, primer 19, primer 21, primer 23, primer 25, primer 27, primer 29, primer 31, primer 33, primer 35, primer 37, primer 39, primer 41, primer 43, primer 45, primer 47, primer 49 and primer 51 are all fluorescently labeled.

4. The primer combination of claim 3, wherein: the 5' ends of primer 1, primer 3, primer 5, primer 7, primer 9 and primer 11 were labeled with FAM; the 5' ends of primer 13, primer 15, primer 17, primer 19, primer 21, primer 23 and primer 25 were labeled with HEX; the 5' ends of primer 27, primer 29, primer 31, primer 33, primer 35 and primer 37 were labeled with TAMRA; the 5' -ends of primer 39, primer 41, primer 43, primer 45, primer 47, primer 49 and primer 51 were labeled with ROX.

5. A multiplex amplification system based on Y-STR loci comprising the primer combination of any one of claims 1 to 4;

the Y-STR locus includes DYS460, DYS389I/II, DYS390, DYS533, DYS392, DYS518, DYS508, DYS437, DYS458, DYS385ab, GATA-H4, DYS576, DYS643, DYS456, DYS391, DYS447, DYS438, DYS448, DYF387S1, DYS393, DYS439, DYS19, DYS444, DDYS449, and DYS 481.

6. The multiplex amplification system of claim 5, wherein: the composite amplification system also comprises reagents required for carrying out PCR amplification reaction; the reagents required for carrying out the PCR amplification reaction comprise DNA polymerase, dNTP and Mg ²⁺ At least one of BSA, KCl and Tris.

7. A kit comprising the primer combination of any one of claims 1 to 4.

8. A method for preparing the multiplex amplification system according to claim 5 or 6 or the kit according to claim 7, comprising the step of packaging each primer in the primer combination according to any one of claims 1 to 4 separately.

9. Use of a primer combination according to any one of claims 1 to 4 or a multiplex amplification system according to claim 5 or 6 for the preparation of a kit for STR typing of male individuals.

10. Use of a primer combination according to any one of claims 1 to 4 or a multiplex amplification system according to claim 5 or 6 for STR typing of male individuals; the use is for the diagnosis and treatment of non-diseases.

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