CN112176068B - Composite amplification system based on 29Y-STR loci and primer combination used by same - Google Patents
Composite amplification system based on 29Y-STR loci and primer combination used by same Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of forensic medicine, and particularly relates to a multiplex amplification system based on 29Y-STR loci and a primer combination used by the multiplex amplification system, in particular to a multiplex amplification system based on 17 universal Y-STR loci, 5 rapid mutation Y-STR loci and 7 Chinese population Y-STR loci and a primer combination used by the multiplex amplification system.
Background
The Y chromosome is unique to males, and all male individuals of the same paternal line have the same or similar Y-STR haplotype. Based on the father line genetic characteristics, the created Y-STR family investigation method is a brand new attempt of applying the Y-STR technology to criminal investigation and case solving, and provides a new working idea for the current public security organs to detect and solve cases in time. The Y-STR family inspection method is an inspection method for accurately inspecting the criminals in the family by utilizing technical means such as autosome detection and the like after an inspector carries out effective Y-STR typing on collected biological inspection materials left by the criminals of the male criminals, searching male individuals with the same or similar Y-STR typing as field material evidence in a certain regional range or a Y-STR database, inspecting the family where the criminals are located. At present, the Y-STR family investigation becomes an important means for case investigation, and particularly provides very important directive clues for case investigation in some cases which are in familial residences and occur in remote areas with less personnel flow. The Y-STR detection reagent is used for detecting male genetic characteristics and plays an important role in general case detection and Y family investigation.
Disclosure of Invention
The invention aims to perform STR typing on male individuals.
The primer combination is protected firstly, and can comprise a primer 1, a primer 2, a primer 3, a primer 4, a primer 5, a primer 6, a primer 7, a primer 8, a primer 9, a primer 10, a primer 11, a primer 12, a primer 13, a primer 14, a primer 15, a primer 16, a primer 17, a primer 18, a primer 19, a primer 20, a primer 21, a primer 22, a primer 23, a primer 24, a primer 25, a primer 26, a primer 27, a primer 28, a primer 29, a primer 30, a primer 31, a primer 32, a primer 33, a primer 34, a primer 35, a primer 36, a primer 37, a primer 38, a primer 39, a primer 40, a primer 41, a primer 42, a primer 43, a primer 44, a primer 45, a primer 46, a primer 47, a primer 48, a primer 49, a primer 50, a primer 51 and a 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.
The primer combination specifically comprises a primer 1, a primer 2, a primer 3, a primer 4, a primer 5, a primer 6, a primer 7, a primer 8, a primer 9, a primer 10, a primer 11, a primer 12, a primer 13, a primer 14, a primer 15, a primer 16, a primer 17, a primer 18, a primer 19, a primer 20, a primer 21, a primer 22, a primer 23, a primer 24, a primer 25, a primer 26, a primer 27, a primer 28, a primer 29, a primer 30, a primer 31, a primer 32, a primer 33, a primer 34, a primer 35, a primer 36, a primer 37, a primer 38, a primer 39, a primer 40, a primer 41, a primer 42, a primer 43, a primer 44, a primer 45, a primer 46, a primer 47, a primer 48, a primer 49, a primer 50, a primer 51 and a primer 52.
In any of the primer combinations described above, the molar ratio 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 may be 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 are all fluorescently labeled.
In any 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 invention also discloses a Y-STR locus multiplex amplification system, which can comprise any one of the primer combinations;
the Y-STR locus may include DYS460, DYS389I/II, DYS390, DYS533, DYS392, DYS518, DYS508, DYS437, DYS458, DYS385ab, GATA-H4, DYS576, DYS643, DYS456, DYS391, DYS447, DYS438, DYS448, DYS 387S1, DYS393, DYS635, DYS439, DYS19, DYS444, DDYS449, and DYS 481.
DYS449, DYS518, DYS576 and DYF387S1 are all rapidly mutating Y-STR loci.
The composite amplification system can specifically consist of any one of the primer combinations.
In any of the above multiplex amplification systems, the concentration of primer 1 and primer 2 in the multiplex amplification system may be 35 mM. The concentration of primer 3, primer 4, primer 5 and primer 6 in the multiplex 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 multiplex amplification system may be 100 mM. The concentration of primer 11, primer 12, primer 25, primer 26, primer 37 and primer 38 in the multiplex amplification system may be 125 mM. The concentration of primer 13 and primer 14 in the multiplex amplification system may be 70 mM. The concentration of primer 15, primer 16, primer 31 and primer 32 in the multiplex amplification system may be 55 mM. The concentration of primer 17 and primer 18 in the multiplex 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 multiplex amplification system may be 90 mM. The concentration of primer 27, primer 28, primer 43 and primer 44 in the multiplex amplification system may be 50 mM. The concentration of primer 29, primer 30, primer 41 and primer 42 in the multiplex amplification system may be 65 mM. The concentration of primer 39 and primer 40 in the multiplex amplification system may be 45 mM. The concentration of primer 51 and primer 52 in the multiplex amplification system may be 150 mM.
In the above composite amplification system, the composite amplification system may further comprise reagents required for performing a PCR amplification reaction; the "reagents required for performing a PCR amplification reaction" does not include primers required for a PCR amplification reaction.
The "reagents required for carrying out PCR amplification reaction" may include DNA polymerase, dNTP, Mg 2 At least one of + BSA, KCl and Tris.
The concentration of the DNA polymerase in the multiplex amplification system may be 0.1U/. mu.L. The concentration of the dNTPs in the multiplex amplification system can be 200. mu.M (i.e., the concentration of dATP, dTTP, dCTP, and dGTP is 200. mu.M). The Mg 2 + may be at a concentration of 1.5mM in said multiplex amplification system. The concentration of the BSA in the multiplex amplification system may be 0.8 mg/mL. The concentration of the KCl in the multiplex amplification system can be 50 mM. The concentration of the Tris in the multiplex amplification system may be 10 mM.
The composite amplification system can specifically comprise any one of the primer combinations and reagents required for carrying out PCR amplification reaction.
A kit containing any of the primer combinations also belongs to the protection scope of the invention. The kit is used for STR typing of male individuals.
The preparation method of any one of the above composite amplification systems or the kit containing any one of the above primer combinations also belongs to the protection scope of the invention.
The method for preparing the multiplex amplification system or the kit containing the primer combination may comprise the step of packaging each primer in any one of the primer combinations separately.
The following X1) or X2) also belong to the scope of the present invention.
X1) any one of the primer combinations or the multiplex amplification system, and the application thereof in preparing a kit for STR typing of male individuals.
X2) any one of the primer combinations or the composite amplification system, and the application thereof in STR typing of male individuals.
Any one of the STR typing may be Y-STR typing.
Any of the above male individuals may be chinese male individuals.
The composite amplification system provided by the invention is adopted to carry out STR typing on a sample I (9948 human genome DNA) or a sample II (a human (known as male) blood collection card). The result shows that the first sample and the second sample both obtain complete STR typing, and the peaks are sharp and clear, the balance is good, and no Pull-up peak, stutter band and non-specific amplification product appear. 9948 results of human genome DNA and Ladder show that the composite amplification system has correct typing results of 29Y-STR loci (comprising 20 core loci, 8 preferred loci and 1 alternative locus specified by public security department of criminal investigation) shown in Table 1, and can completely meet the requirements of forensic Y-STR inspection. Therefore, the composite amplification system provided by the invention can be used for amplifying 29Y-STR loci (5 of which are rapid mutation Y-STR loci and 7 of which are Chinese population Y-STR loci) shown in Table 1, 29 different allele fragments can be obtained at most by one-time amplification of the composite amplification system, and the amplification efficiency is very 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 provided by the invention 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 identification capability of the system. In addition, the composite amplification system is suitable for 3130, 3500, 3730 and other types of sequencers, all gene loci are less than 470bp, and the composite amplification system has good adaptability to degradation detection materials. The invention has important application value.
Drawings
FIG. 1 is a DNA detection map of sample one.
FIG. 2 is a DNA detection map of sample two.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
9948 human genomic DNA is available from Promaga.
Example 1 preparation of a multiplex amplification System based on 29Y-STR loci
Screening of the first, Y-STR loci
The inventor of the invention obtains a large number of candidate Y-STR loci by consulting the literature; then, searching by using the reported primer information of each locus on a UCSC website (the website is http:// genome. UCSC. edu /) by using the research result of the human genome plan to obtain a corresponding sequence; searching and inquiring the sequence in GenBank through BLAST (website: https:// blast.ncbi.nlm.nih.gov/blast.cgi), then sorting and systematically researching the sequence information, allelic length, distribution and the like of the core and flanking regions of the existing Y-STR locus, and focusing on the Y-STR locus with the core sequence of 4-6 base repetition. And finally screening to obtain 29Y-STR loci including 17 general Y-STR loci, 5 rapid mutation Y-STR loci and 7Y-STR loci of Chinese population by comprehensively considering the technical indexes of copy number, mutation rate, repetitive unit characteristics, sequence structure complexity and the like of each locus, compatibility with other commercial kits and the like.
The 17 universal Y-STR loci are respectively: DYS19, DYS385ab, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, GATA-H4, DYS438, and DYS 439.
5 rapidly mutating Y-STR loci: DYS449, DYS518, DYS576, DYF387S 1.
7 Chinese population Y-STR loci: DYS460, DYS447, DYS444, DYS508, DYS533, DYS643, DYS 481.
It should be noted that there is no obvious difference in the length of the amplified fragments of DYS385ab and DYF387S1, and they are conventionally distinguished by a/b, i.e. DYS385ab is divided into DYS385a and DYS385b, DYF387S1 is divided into DYF387S1a and DYF387S1 b; the amplified fragments of DYS389I/II were significantly different in length and were customarily separated by I/II, i.e., DYS389I/II was divided into DYS389I and DYS389 II.
Preparation of composite amplification system based on 29Y-STR loci
1. The method comprises the steps of preliminarily screening suitable fluorescent dyes according to the range of the emission wavelength of each fluorescent dye, the strength of a fluorescent signal and the like, testing the stability of the fluorescent dyes through a large number of experiments, finally obtaining five-color fluorescent dyes which are not interfered with each other and have stable quality, and realizing five-color fluorescence detection.
2. According to the allelic gene composition, range and amplified fragment length (all less than 470bp) of each locus, fluorescent markers with different colors are reasonably selected to distinguish each locus in the fragment overlapping region, and then a composite amplification system based on 29Y-STR loci is prepared. The method comprises the following specific steps:
(1) primers for amplifying each locus were artificially synthesized and then subjected to PCR amplification to obtain specific primers for each locus.
(2) Synthesizing the amplification conditions of a single locus, selecting a proper amplification program, carrying out composite amplification on the primers with the same color fluorescence markers, and carrying out composite amplification on the primers with different color fluorescence markers after confirmation. During multiplex amplification, each locus was observed for the presence of specific and non-specific products. If non-specific hybrid peaks occur between primers of different loci, the primers need to be designed and synthesized again by finding out the loci one by one. In addition, primer dimers are formed between primers at different loci, which reduces the amplification efficiency of the primers and requires redesign and synthesis of primers.
(3) The complex amplification system is repeatedly tested and modified, the genome DNA of the male is amplified by the complex amplification system, whether the loci overlap or not is observed, because the amplification product is designed according to the theoretical size, the migration rate is faster or slower in the actual process due to the inconsistent sequence structure of the amplification product, the theoretical size is not consistent with the actual size, the adjacent loci are overlapped or too close to each other, and the subsequent analysis is influenced. Once this occurs, the locus primer needs to be redesigned and go through steps (1) and (2), finally obtaining a multiplex amplification system satisfying the conditions.
(4) Leveling of the multiplex amplification system. The method specifically comprises the following steps: during primary composite amplification, the concentration of each primer in a reaction system is 100 mM; the concentration of each primer pair is adjusted according to the reaction result.
(5) And optimizing the parameters of the composite amplification reaction. 9948 human genome DNA is used to adjust key components and links in the reaction system, such as optimal reaction system, amplification cycle number, annealing temperature, extension time, amount of hot-start DNA polymerase, primer amount, buffer concentration, etc.
Through the steps, a composite amplification system based on 29Y-STR loci is obtained. The composite amplification system consists of DNA polymerase, dNTP and Mg 2+ BSA, KCl, Tris and a primer mixture; the primer mixture is formed by mixing 52 primers; the names, nucleotide sequences, alleles and amplified loci of the 52 primers are detailed in Table 1, columns 1-5. In the multiplex amplification system, the concentration of DNA polymerase is 0.1U/. mu.L, the concentration of dNTP is 200. mu.M (i.e., the concentrations of dATP, dTTP, dCTP and dGTP are all 200. mu.M), and Mg 2+ Was 1.5mM, BSA was 0.8mg/mL, KCl was 50mM, Tris was 10mM, and the concentrations of 52 primers are shown in column 6 of Table 1.
TABLE 1
Note: FAM indicates FAM labeling at the 5' end; HEX indicates HEX labeling of the 5' terminus; TAMRA indicates TAMRA labeling at the 5' end; ROX indicates ROX labeling at the 5' end.
3. Preparing the composite amplification system based on 29Y-STR loci.
Example 2 application of multiplex amplification System based on 29Y-STR loci
The Typer500 internal standard is a product of a public security department material evidence authentication center. The deionized formamide is a product of ABI company under the catalog number 4311320. The ABI3130xl genetic analyzer is a product of ABI. The human (known as male) blood collection card is provided by the construction of a daily DNA database at the material evidence identification center of the Ministry of public Security.
The sample to be detected is a sample I or a sample II.
A first sample: 1ng 9948 human genomic DNA.
Sample two: a human (known as male) blood collection card was taken and a disk was punched out with a 0.5mm hand-held punch.
1. And (3) adding 10 mu L of the composite amplification system prepared in the step two in the embodiment 1 into a sample to be detected for PCR amplification to obtain a PCR amplification product.
The reaction procedure is as follows: pre-denaturation at 95 ℃ for 11 min; denaturation at 94 deg.C for 30s, annealing at 59 deg.C for 2min, extension at 72 deg.C for 1min, and 28 times of circulation; extending for 60min at 60 ℃; storing at 4 ℃.
2. After completion of step 1, 10. mu.L of the sample mixture and 1. mu.L of the PCR amplification product were mixed uniformly to obtain a reaction solution.
The loading mixture was mixed with 10. mu.L of Typer500 internal standard and 1000. mu.L of deionized formamide.
3. And (3) after the step 2 is finished, taking the reaction solution, denaturing at 95 ℃ for 5min, quickly transferring to-20 ℃ and standing for 5min, and then carrying out capillary electrophoresis detection by using an ABI3130xl genetic analyzer to obtain a DNA detection map. The electrophoresis conditions are as follows: the sample injection voltage is 1.2kV, and the sample injection time is 30 s.
The result of the detection of sample one is shown in fig. 1.
The detection result of sample two is shown in FIG. 2.
The result shows that the first sample and the second sample both obtain complete STR typing, and the peaks are sharp and clear, the balance is good, and no Pull-up peak, stutter band and non-specific amplification product appear. 9948 the results of the human genome DNA also show that the composite amplification system prepared in example 1 has correct typing results for 29Y-STR loci shown in Table 1, and can completely meet the requirements of forensic Y-STR tests.
The composite amplification system provided by the invention can be used for amplifying 29Y-STR loci (5 of which are rapid mutation Y-STR loci and 7 of which are Chinese population Y-STR loci) shown in Table 1, 29 different allele fragments can be obtained at most by one-time amplification of the composite amplification system, and the amplification efficiency is very 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 provided by the invention 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 identification capability of the system.
<110> material evidence identification center of public security department
<120> a multiplex amplification system based on 29Y-STR loci and primer combination used by same
<160> 52
<170> PatentIn version 3.5
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<210> 19
<211> 21
<212> DNA
<213> Artificial sequence
<400> 19
gagctagaca ccatgccaaa c 21
<210> 20
<211> 25
<212> DNA
<213> Artificial sequence
<400> 20
attccaatta catagtcctc ctttc 25
<210> 21
<211> 23
<212> DNA
<213> Artificial sequence
<400> 21
ctttcagcac atcacttgta tcc 23
<210> 22
<211> 23
<212> DNA
<213> Artificial sequence
<400> 22
tttcctctga tggtgaagta atg 23
<210> 23
<211> 25
<212> DNA
<213> Artificial sequence
<400> 23
aaaagttatt gactaaatgg gatgc 25
<210> 24
<211> 24
<212> DNA
<213> Artificial sequence
<400> 24
gggaacatag tcaaaccata tcag 24
<210> 25
<211> 23
<212> DNA
<213> Artificial sequence
<400> 25
atgcctggtt aaactactgt gcc 23
<210> 26
<211> 24
<212> DNA
<213> Artificial sequence
<400> 26
ggaattaaat aatggcattg gtag 24
<210> 27
<211> 24
<212> DNA
<213> Artificial sequence
<400> 27
ggaccttgtg ataatgtaag atag 24
<210> 28
<211> 23
<212> DNA
<213> Artificial sequence
<400> 28
attagggttc tctagaggga cag 23
<210> 29
<211> 27
<212> DNA
<213> Artificial sequence
<400> 29
ctattcattc aatcatacac ccatatc 27
<210> 30
<211> 21
<212> DNA
<213> Artificial sequence
<400> 30
ggaataaaat ctccctggtt g 21
<210> 31
<211> 21
<212> DNA
<213> Artificial sequence
<400> 31
gttatctctg cctttctgga c 21
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence
<400> 32
gtcacagcat ggcttggttt t 21
<210> 33
<211> 22
<212> DNA
<213> Artificial sequence
<400> 33
ctgatgcaag aaagattcac tg 22
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence
<400> 34
gtggcagacg cctataatcc 20
<210> 35
<211> 22
<212> DNA
<213> Artificial sequence
<400> 35
tgtcaaagag cttcaatgga ga 22
<210> 36
<211> 22
<212> DNA
<213> Artificial sequence
<400> 36
cctgtgttgg agaccttttc tt 22
<210> 37
<211> 24
<212> DNA
<213> Artificial sequence
<400> 37
gctagattcc attttacccc taac 24
<210> 38
<211> 22
<212> DNA
<213> Artificial sequence
<400> 38
atgcgagtct cactagctgg tc 22
<210> 39
<211> 24
<212> DNA
<213> Artificial sequence
<400> 39
gtcattccta atgtggtctt ctac 24
<210> 40
<211> 24
<212> DNA
<213> Artificial sequence
<400> 40
tgaggtatgt ctcatagaaa agac 24
<210> 41
<211> 21
<212> DNA
<213> Artificial sequence
<400> 41
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 atagtg 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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