CN110777211B - Composite amplification system based on Y-STR locus and Y-indel locus and primer combination used by same - Google Patents
Composite amplification system based on Y-STR locus and Y-indel locus and primer combination used by same Download PDFInfo
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Abstract
The invention discloses a composite amplification system based on a Y-STR locus and a Y-indel locus and a primer combination used by the same. The primer combination provided by the invention consists of 74 DNA molecules shown in a sequence 1 to a sequence 74 in a sequence table. The primer combination is adopted to construct a composite amplification system, then male STR typing is carried out, the detected polymorphism is high, and the method has important identification value in the aspects of paternal relationship identification, family investigation and the like. The invention has important application value.
Description
Technical Field
The invention belongs to the technical field of forensic medicine, and particularly relates to a composite amplification system based on Y-STR loci and Y-indel loci and a primer combination used by the composite amplification system, in particular to a composite amplification system based on 38Y-STR loci and 3Y-indel loci and a primer combination used by the composite amplification system.
Background
Short Tandem Repeat (STR) belongs to the second generation genetic marker, and is widely applied to the fields of population genetics and forensic medicine due to the characteristics of small repeat unit, high mutation rate, wide distribution in genome and the like. In the human genome, there is one STR site approximately every 15-20kb, which accounts for 10% of the total human genome. These STRs usually consist of 2-6 bases and are composed of tandem repeat units, and due to the difference in repeat units and repeat times, they show great variability among people of different ethnic groups and regions, and are manifested as genetic polymorphisms.
The Y-STR genetic marker is an STR genetic marker distributed on human Y chromosome, is an effective means for testing and identifying in the field of court science at present, and is an important supplement of autosomal STR, mitochondrial genetic markers and the like. Because the Y chromosome is peculiar to the male individual, most (more than 95 percent) of the Y chromosome is a non-recombinant region, the Y-STR presents the paternal inheritance characteristic and plays an important role in the aspects of paternal inheritance relationship identification, mixed-spot male individual inspection and the like. Meanwhile, by utilizing the characteristic that the Y-STR is relatively conserved in one family, the family of the important region can be subjected to Y-STR typing inspection to carry out family investigation, so that the investigation personnel are assisted to quickly lock the family of the suspect and the progress of the case is accelerated. In recent years, the Y-STR technology has helped to break down a large number of hit-case scenarios.
With the development of Y-STR technology, the amplification and inspection system related to the Y-STR at home and abroad is continuously improved, and the reagent products are continuously enriched. Initially, the Y-STR amplification detection system can only detect a few Y-STR loci. Subsequently, the number of loci in the reagent System is increased continuously, and the detection efficiency is enhanced continuously, for example, the Promega company firstly provides a PowerPlex Y System containing 12Y-STR loci, and then develops a PowerPlex Y23 System capable of detecting 23Y-STR loci; the ABI company provides a Yfiler kit containing 17Y-STR loci, and then develops Yfiler Plus capable of detecting 27Y-STR loci; in China, DNATYPERY series reagents are independently developed by the material evidence identification center of the Ministry of public Security, and Y-STR detection reagents are continuously proposed by Texas China Union biotechnology Limited and Suzhou Ministry of Japan and GenBank technology Limited, so that six-color fluorescent amplification detection products containing more than 30Y-STR loci are available at present. The product can play a certain identification role in the inspection materials for male and female component mixing or multiple male component mixing, and can assist in searching the family of the criminal suspect, thereby greatly reducing the investigation range. However, existing kit products show deficiencies in both locus number and locus mutation rate, and thus in some case applications, male individuals cannot be effectively distinguished. Based on this, the Y-STR test reagent product should contain more loci, and in particular should contain 20Y-STR core loci (e.g., DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, Y GATA H4, DYS438, DYS439, DYS460, DYS533, and DYS 576) and 15 preferred loci (e.g., DYS449, DYS518, DYS 387S1, DYS627, DYS570, DYS447, DYS444, DYS549, DYS557, DYS643, DYS481, DYS596, DYS527 a/b) selected by the ministry of shandong for the construction of the Y database, and DYS593 and DYS645 selected by the ministry of japan for the Y database, so as to achieve a greater efficacy in the test system and a greater practical application.
Disclosure of Invention
The present invention is directed to STR typing of 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, a primer 52, a primer 53, a primer 54, a primer 55, a primer 56, a primer 57, a primer 58, a primer 59, a primer 60, a primer 61, a primer 62, a primer 63, a primer 64, a primer 65, a primer 66, a primer 67, a primer 68, a 69, a primer 70, a primer 71, a primer 74, a primer and a 73;
primer 1 can be SEQ ID NO:1, a single-stranded DNA molecule;
primer 2 can be SEQ ID NO: 2;
primer 3 can be SEQ ID NO:3, a single-stranded DNA molecule;
primer 4 can be SEQ ID NO:4, a single-stranded DNA molecule;
primer 5 can be SEQ ID NO:5, a single-stranded DNA molecule;
primer 6 can be SEQ ID NO: 6;
primer 7 can be SEQ ID NO: 7;
primer 9 can be SEQ ID NO:9, a single-stranded DNA molecule;
primer 11 can be SEQ ID NO: 11;
primer 12 can be SEQ ID NO: 12;
primer 16 can be SEQ ID NO: 16;
primer 17 can be SEQ ID NO: 17;
primer 19 can be SEQ ID NO: 19;
primer 22 can be SEQ ID NO: 22;
primer 23 can be SEQ ID NO: 23;
primer 25 can be SEQ ID NO: 25;
primer 26 can be SEQ ID NO: 26;
primer 27 can be SEQ ID NO:27, a single-stranded DNA molecule;
primer 28 may be SEQ ID NO: 28;
primer 29 can be SEQ ID NO: 29;
the primer 30 may be SEQ ID NO: 30;
primer 31 can be SEQ ID NO: 31;
primer 32 can be SEQ ID NO:32, a single-stranded DNA molecule;
primer 33 may be SEQ ID NO: 33;
primer 34 can be SEQ ID NO:34, a single-stranded DNA molecule;
primer 35 can be SEQ ID NO: 35;
primer 36 can be SEQ ID NO: 36;
primer 37 may be SEQ ID NO: 37;
primer 38 can be SEQ ID NO: 38;
primer 39 can be SEQ ID NO: 39;
primer 40 can be SEQ ID NO: 40;
primer 41 can be SEQ ID NO: 41;
primer 42 can be SEQ ID NO: 42;
primer 43 can be SEQ ID NO: 43;
primer 44 may be SEQ ID NO:44, a single-stranded DNA molecule;
primer 45 can be SEQ ID NO:45, a single-stranded DNA molecule;
primer 46 may be SEQ ID NO: 46;
primer 47 can be SEQ ID NO:47, a single-stranded DNA molecule;
primer 48 can be SEQ ID NO:48, a single-stranded DNA molecule;
primer 49 can be SEQ ID NO:49, or a single-stranded DNA molecule thereof;
primer 50 can be SEQ ID NO: 50;
primer 51 may be SEQ ID NO: 51;
primer 52 can be SEQ ID NO: 52;
primer 53 may be SEQ ID NO:53, a single-stranded DNA molecule;
primer 54 can be SEQ ID NO: 54;
primer 55 can be SEQ ID NO: 55;
primer 56 can be SEQ ID NO: 56;
primer 57 can be SEQ ID NO: a single-stranded DNA molecule as set forth in 57;
primer 58 can be SEQ ID NO: 58;
primer 59 can be SEQ ID NO: 59;
primer 60 can be SEQ ID NO: 60;
primer 61 can be SEQ ID NO: 61;
primer 62 can be SEQ ID NO: 62;
primer 63 may be SEQ ID NO: 63;
primer 64 can be SEQ ID NO: 64;
primer 65 may be SEQ ID NO: 65;
primer 66 can be SEQ ID NO: 66;
primer 67 may be SEQ ID NO:67, a single stranded DNA molecule;
primer 68 can be SEQ ID NO: 68;
primer 69 can be SEQ ID NO: 69;
primer 70 can be SEQ ID NO: 70;
primer 71 may be SEQ ID NO: 71;
primer 72 can be SEQ ID NO: 72;
primer 73 can be SEQ ID NO: 73;
primer 74 may be SEQ ID NO:74 under stringent conditions.
The primer combination may specifically 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, primer 52, primer 53, primer 54, primer 55, primer 56, primer 57, primer 58, primer 59, primer 60, primer 61, primer 62, primer 63, primer 64, primer 65, primer 66, primer 67, primer 68, primer 69, primer 70, primer 74, primer 72, and primer 73.
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, primer 52, primer 53, primer 54, primer 55, primer 56, primer 57, primer 58, primer 59, primer 60, primer 61, primer 62, primer 63, primer 64, primer 65, primer 66, primer 67, primer 68, primer 69, primer 70, primer 74, primer 72, primer 74, and primer 72 may be: 150:70:70:140:140:180:180:170:170:300:300:160:160:130:130:130:130:120:120:80:80:90:90:140:140:180:180:100:100:150:150:180:180:210:210:120:120:200:200:210:210:160:160:400:400:180:180:120:120:120:120:200:200:200:200:250:250:400:400:150:150:230:230:130:130:190:190:210:210:140:140:300:300.
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, primer 51, primer 53, primer 55, primer 57, primer 59, primer 61, primer 63, primer 65, primer 67, primer 69, primer 71 and primer 73 may be labeled with a fluorescent label.
In any of the primer combinations described above, primer 1, primer 3, primer 5, primer 7, primer 9, primer 11, primer 13, primer 15 and primer 17 may be labeled with FAM. Primer 19, primer 21, primer 23, primer 25, primer 27, primer 29 and primer 31 may be labeled with HEX. Primer 33, primer 35, primer 37, primer 39, primer 41, primer 43 and primer 45 may be labeled with TAMRA. Primer 47, primer 49, primer 51, primer 53, primer 55, primer 57 and primer 59 may be labeled with ROX. Primer 61, primer 63, primer 65, primer 67, primer 69, primer 71 and primer 73 can be labeled with SID. The 5' ends of primer 1, primer 3, primer 5, primer 7, primer 9, primer 11, primer 13, primer 15 and primer 17 may be labeled with FAM. The 5' ends of primer 19, primer 21, primer 23, primer 25, primer 27, primer 29 and primer 31 may be labeled with HEX. The 5' ends of primer 33, primer 35, primer 37, primer 39, primer 41, primer 43 and primer 45 may be labeled with TAMRA. The 5' ends of primer 47, primer 49, primer 51, primer 53, primer 55, primer 57 and primer 59 may be labeled with ROX. The 5' ends of primer 61, primer 63, primer 65, primer 67, primer 69, primer 71 and primer 73 may be labeled with SID.
The invention also discloses a composite amplification system based on the Y-STR locus and the Y-Indels locus, which can comprise any one of the primer combinations;
the Y-STR locus may comprise DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, Y _ GATA _ H4, DYS438, DYS439, DYS460, DYS533, DYS576, DYS449, DYS518, dysf 387S1, DYS627, DYS570, DYS447, DYS444, DYS549, DYS557, DYS643, DYS481, DYS596, DYS527a/b, DYS508, DYS593, and DYS645;
the Y-Indels locus may comprise rs771783753, rs199815934 and rs759551978.
The Y-STR locus may specifically consist of DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, Y _ GATA _ H4, DYS438, DYS439, DYS460, DYS533, DYS576, DYS449, DYS518, dysf 387S1, DYS627, DYS570, DYS447, DYS444, DYS549, DYS557, DYS643, DYS481, DYS596, DYS527a/b, DYS508, DYS593, and DYS645.
The Y-Indels locus may specifically consist of rs771783753, rs199815934 and rs759551978.
DYS576, DYS449, DYS518, DYF387S1, DYS627 and DYS570 were rapidly mutating the Y-STR locus.
The 41 loci (i.e., 38Y-STR loci and 3Y-Indels loci) comprise 20Y-STR core loci and 15 preferred loci selected by the public security department for construction of a Y database, and also comprise DYS593 and DYS645 selected by the public security organization of Shandong province for construction of the Y database.
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, primer 2, primer 31, primer 32, primer 61 and primer 62 in the multiplex amplification system may be 150mM. The concentration of each of primer 3 and primer 4 in the multiplex amplification system may be 70mM. Primer 5, primer 6, primer 25, primer 26, primer 71 and primer 72 may each be present in the multiplex amplification system at a concentration of 140mM. The concentrations of primer 7, primer 8, primer 27, primer 28, primer 33, primer 34, primer 47 and primer 48 in the multiplex amplification system were all 180mM. The concentration of each of primer 9 and primer 10 in the multiplex amplification system may be 170mM. The concentration of primer 11, primer 12, primer 73 and primer 74 in the multiplex amplification system may be 300mM each. The concentration of each of primer 13, primer 14, primer 43 and primer 44 in the multiplex amplification system may be 160mM. The concentration of primer 15, primer 16, primer 17, primer 18, primer 65 and primer 66 in the multiplex amplification system may be 130mM each. The concentration of each of primer 19, primer 20, primer 37, primer 38, primer 49, primer 50, primer 51 and primer 52 in the multiplex amplification system may be 120mM. The concentration of each of primer 21 and primer 22 in the multiplex amplification system may be 80mM. The concentration of each of the primer 23 and the primer 24 in the multiplex amplification system may be 90mM. The concentration of each of the primer 29 and the primer 30 in the multiplex amplification system may be 100mM. The concentration of primer 35, primer 36, primer 41, primer 42, primer 69 and primer 70 in the multiplex amplification system may each be 210mM. The concentration of each of primer 39, primer 40, primer 53, primer 54, primer 55 and primer 56 in the multiplex amplification system may be 200mM. The concentration of primer 45, primer 46, primer 59, and primer 60 in the multiplex amplification system may each be 400mM. The concentration of each of the primer 57 and the primer 58 in the multiplex amplification system may be 250mM. The concentration of each of the primers 63 and 64 in the multiplex amplification system may be 230mM. The concentration of each of primer 67 and primer 68 in the multiplex amplification system may be 190mM.
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+ The concentration in the multiplex amplification system may be 1.5mM. The concentration of the BSA in the multiplex amplification system may be 0.8mg/mL. The concentration of the KCl in the multiplex amplification system can be 50mM. The concentration of Tris in the multiplex amplification system may be 10mM.
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 protection of the present invention.
X1) any one of the primer combinations or any one of the multiplex amplification systems, and the application thereof in preparing a kit for STR typing of male individuals.
X2) the primer combination or the multiplex amplification system, and is applied to 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 the sample I (9948 human genome DNA standard) or the sample II (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 the human genome DNA standard also show that the typing results of the composite amplification system for 38Y-STR loci and 3Y-Indels loci shown in Table 1 are correct, and the requirements of forensic Y-STR inspection can be completely met. Therefore, the composite amplification system provided by the invention can be used for amplifying 38Y-STR loci (7 of which are rapid mutation Y-STR loci) and 3Y-Indels loci, and can obtain 41 different allelic fragments at most by one-time amplification, so that the amplification efficiency is very high. Meanwhile, the multiplex amplification system comprises 7Y-STR loci with rapid mutation, and the detection efficiency is very strong. The composite amplification system provided by the invention has important application value in the aspects of paternal genetic relationship identification, family investigation and the like.
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.
Example 1 preparation of a multiplex amplification System based on 38Y-STR loci and 3Y-indel loci
1. Screening of Y-STR locus and Y-indel locus
The inventor obtains a large number of candidate Y-STR loci and Y-indel 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 core and flanking region sequence information, allele length, distribution and the like of the existing Y-STR locus and Y-indel locus, and focusing on the Y-STR locus with the core sequence of 4-5 base repetition and the Y-indel locus with the insertion fragment of not less than 3 bp. And finally screening 38Y-STR loci and 3Y-indel loci by comprehensively considering the technical indexes such as copy number, mutation rate, repetitive unit characteristics, sequence structure complexity and the like of each locus and the compatibility with other commercial kits.
The 38Y-STR loci are respectively: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, Y _ GATA _ H4, DYS438, DYS439, DYS460, DYS533, DYS576, DYS449, DYS518, DYF387S1, DYS627, DYS570, DYS447, DYS444, DYS549, DYS557, DYS481, DYS596, DYS527a/b, DYS508, DYS593, DYS645. Wherein DYS576, DYS449, DYS518, DYF387S1, DYS627 and DYS570 are rapid mutation Y-STR loci.
The 3Y-indel loci are: rs771783753, rs199815934 and rs759551978.
It should be noted that there is no obvious difference in the length of the amplification fragments of DYS385a/b, DYS527a/b and DYF387S1, and they are conventionally distinguished by a/b, i.e. DYS385a/b is divided into DYS385a and DYS385b, DYS527a/b is divided into DYS527a and DYS527b, DYF387S1 is divided into DYF387S1a and DYF387S1b; the amplified fragments of DYS389I/II have significant differences in length and are customarily distinguished by I/II, i.e., DYS389I/II is divided into DYS389I and DYS389II.
The 41 loci comprise 20Y-STR core loci and 15 preferred loci selected by the Ministry of public Security for Y database construction, and also comprise DYS593 and DYS645 selected by the Ministry of public Security of Shandong province for Y database construction.
2. Preparation of composite amplification system based on 38Y-STR loci and 3Y-Indels loci
1. The appropriate fluorescent dyes are preliminarily screened according to the range of the emission wavelength of each fluorescent dye, the intensity of a fluorescent signal and the like, then the stability of the fluorescent dyes is tested through a large number of experiments, and finally the six-color fluorescent dyes which are not interfered with each other and have stable quality are obtained, so that the six-color fluorescent detection is realized.
2. According to the allelic gene composition, range and amplified fragment length 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 38Y-STR loci and 3Y-Indels 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 single locus, selecting a proper amplification program, carrying out composite amplification on the primers with the same color fluorescence label, and carrying out composite amplification on the primers with different color fluorescence labels 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 of 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) to finally obtain 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 100mM; the concentration of each primer pair is adjusted according to the reaction result.
(5) And optimizing the parameters of the multiplex amplification reaction. 9948 human genome DNA standard is used to regulate the key components and links in the reaction system, such as amplification cycle number, annealing temperature, extension time, hot start DNA polymerase dosage, primer amount, buffer concentration, etc.
Through the steps, a composite amplification system based on 38Y-STR loci and 3Y-Indels 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 74 primers; the information on the names, nucleotide sequences, alleles and amplified loci of the 74 primers are detailed in columns 1-5 of Table 1. 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+ Has a concentration of 1.5mM, a concentration of BSA of 0.8mg/mL, a concentration of KCl of 50mM, a concentration of Tris of 10mM, and 74The concentrations of the strip primers are shown in column 6 of Table 1.
TABLE 1
Note: FAM indicates FAM labeling of the 5' end; HEX indicates HEX labeling of the 5' terminus; TAMRA indicates TAMRA labeling at the 5' end; ROX indicates that the 5 'end is ROX-tagged and SID indicates that the 5' end is SID-tagged.
3. And preparing the composite amplification system based on 38Y-STR loci and 3Y-Indels loci.
Example 2 application of multiplex amplification System based on 38Y-STR loci and 3Y-indel 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 catalog number 4311320.ABI3130xl genetic Analyzer is a product of ABI company. 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:
sample one, 1ng9948 human genome DNA standard;
sample two: a blood collection card from a human (known as a male) was taken and a circular piece was punched out with a 0.5mm hand-held punch.
1. And (3) adding a sample to be detected into 10 mu L of the composite amplification system prepared in the step two in the embodiment 1 for PCR amplification to obtain a PCR amplification product.
Reaction procedure: pre-denaturation at 95 ℃ for 11min; denaturation at 94 ℃ 30s, annealing at 59 ℃ for 2min, extension at 72 ℃ for 1min,28 cycles; extending for 60min at 60 ℃; storing at 4 deg.C.
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 30s.
The experimental results of sample one are shown in fig. 1 (rs 77 is rs771783753, rs1998 is rs199815934, and rs75955 is rs 759551978).
The experimental results of sample two are shown in fig. 2 (rs 77 is rs771783753, rs1998 is rs199815934, and rs75955 is rs 759551978).
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 standards also show that the composite amplification system prepared in example 1 has correct typing results for the 38Y-STR loci and the 3Y-Indels 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 38Y-STR loci (7 of which are rapid mutation Y-STR loci) and 3Y-indel loci shown in table 1, 41 different allele fragments can be obtained by one-time amplification of the composite amplification system, and the amplification efficiency is very high. Meanwhile, the composite amplification system contains 7 rapidly mutated Y-STR loci, and the detection efficiency is very strong. The composite amplification system provided by the invention can further promote the application of the Y-STR in the aspects of paternal genetic relationship identification, family investigation and the like.
<110> material evidence identification center of public security department
<120> a multiplex amplification system based on Y-STR locus and Y-indel locus and primer combination used by the same
<160> 74
<170> PatentIn version 3.5
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cctctgccta tcatttatta tg 22
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atcaaggcta ttaatcaaaa atac 24
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tgtatccaac tctcatctgt attatct 27
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cgaattatcc ctgagtagca gaa 23
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ccctgcattt tggtacccca ta 22
<210> 10
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gagtgggaga aatggatgac ag 22
<210> 11
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tctatatctg tctattcatc taac 24
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tgtatttatt catgatcagt tc 22
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<400> 13
ttgatgcaat gtaaattcct acag 24
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<211> 22
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<400> 14
catccatgtt gctccaaagg ac 22
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ggaggcttgt cactaatttt tg 22
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ggatatctct cattagatat tc 22
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actccagcct gggcaacaca agtg 24
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<213> Artificial sequence
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tccaaagtgc tttgtggtgt gc 22
<210> 19
<211> 21
<212> DNA
<213> Artificial sequence
<400> 19
gccagcttat tattccttct c 21
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence
<400> 20
gatatgcaga gagtagatat ag 22
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence
<400> 21
gactgagcaa caggaatgaa ac 22
<210> 22
<211> 21
<212> DNA
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<400> 22
cattacaagc atgagccacc a 21
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<211> 21
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<400> 23
ttgagtgcat gcccatccgg t 21
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cagcctgagg aacagaggaa gac 23
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<211> 21
<212> DNA
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<400> 25
gacaccatgc caaacaacaa c 21
<210> 26
<211> 22
<212> DNA
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gtatctattc caattacata gt 22
<210> 27
<211> 22
<212> DNA
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<400> 27
cagcacatca cttgtatcct ag 22
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<211> 22
<212> DNA
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<400> 28
gaccatttcc tctgatggtg aa 22
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<211> 22
<212> DNA
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<400> 29
ggtcatgcca gtaatcccag ta 22
<210> 30
<211> 21
<212> DNA
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ctatcataag aacagcatgg g 21
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<212> DNA
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gctgcaccag tcactgtatt tgc 23
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<212> DNA
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<212> DNA
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ggtctgttgt gggaccttgt ga 22
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ggacagaact aatggaatat c 21
<210> 35
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<212> DNA
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<400> 35
cttgtgtatc tattcattca atc 23
<210> 36
<211> 21
<212> DNA
<213> Artificial sequence
<400> 36
cctggttgca tgcaattgcc a 21
<210> 37
<211> 23
<212> DNA
<213> Artificial sequence
<400> 37
<210> 38
<211> 22
<212> DNA
<213> Artificial sequence
<400> 38
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<211> 22
<212> DNA
<213> Artificial sequence
<400> 39
gagtaacacc attagagcat ca 22
<210> 40
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<400> 40
taggctgatg caggagaatc gct 23
<210> 41
<211> 21
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<400> 41
tatgggagag gcaaggatcc a 21
<210> 42
<211> 21
<212> DNA
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<400> 42
ctcatatttc tggccggtct g 21
<210> 43
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gtgaagtttg cagtgagctg ag 22
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<400> 44
actggagtct gtaagtggct ca 22
<210> 45
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gtctgttcca agtggttcac ag 22
<210> 46
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gctagtgaga gaaagtctcc aat 23
<210> 47
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ggttgtcatt cctaatgtgg tc 22
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ccaaaaaatg aggtatgtct ca 22
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<400> 49
tcttggcttc tcactttgca tag 23
<210> 50
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tgtggaacca gcccaaatat ccat 24
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ggtgttatgg ttttaggtct aac 23
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cctggcttgg aattctttta cc 22
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<400> 53
ctgcacctgg aaatagtggc t 21
<210> 54
<211> 23
<212> DNA
<213> Artificial sequence
<400> 54
tcactatgac tactgagttt ctg 23
<210> 55
<211> 22
<212> DNA
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ggtgtgaacc atttggcatg tt 22
<210> 56
<211> 22
<212> DNA
<213> Artificial sequence
<400> 56
ctggctgtct aagggatcca aa 22
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ggctagagat tcttggagtc tc 22
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gctgtaagct aagattgcac cat 23
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gggtgagtct tttggaaact tg 22
<210> 60
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<212> DNA
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cattgcagat tcttggtcca c 21
<210> 61
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<212> DNA
<213> Artificial sequence
<400> 61
atcctggctg tgtcctccaa g 21
<210> 62
<211> 22
<212> DNA
<213> Artificial sequence
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aatgcagata ttccctatct ac 22
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aaccacttga tttaaggcat cct 23
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gtactgaatg acattaagat gta 23
<210> 66
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gcataagtgg taatgtcccc tt 22
<210> 67
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<212> DNA
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<400> 67
cctactggca ggtgtgattg tga 23
<210> 68
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gtgtttgacc cagtgatatg ttc 23
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gacagcgcag gattccatct a 21
<210> 70
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accatgtgga taatgagcaa atg 23
<210> 71
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<213> Artificial sequence
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ggtggcaatc atagtccata t 21
<210> 72
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<212> DNA
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<400> 72
cccataaatt ccatccaagc tc 22
<210> 73
<211> 22
<212> DNA
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agccacaaca taagtaaggt ag 22
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cagctacttg ggaggataag gc 22
Claims (11)
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, primer 52, primer 53, primer 54, primer 55, primer 56, primer 57, primer 58, primer 59, primer 60, primer 61, primer 62, primer 63, primer 64, primer 65, primer 66, primer 67, primer 68, primer 69, primer 70, primer 71, primer 72, primer 73, and primer 74;
primer 1 is SEQ ID NO:1, a single-stranded DNA molecule;
primer 2 is SEQ ID NO: 2;
primer 3 is SEQ ID NO:3, a single-stranded DNA molecule;
primer 4 is SEQ ID NO:4, a single-stranded DNA molecule;
primer 5 is SEQ ID NO:5, a single-stranded DNA molecule;
primer 6 is SEQ ID NO: 6;
primer 7 is SEQ ID NO: 7;
primer 8 is SEQ ID NO:8, a single-stranded DNA molecule;
primer 9 is SEQ ID NO: 9;
primer 10 is SEQ ID NO:10, a single-stranded DNA molecule;
primer 11 is SEQ ID NO: 11;
primer 12 is SEQ ID NO: 12;
primer 13 is SEQ ID NO:13, a single-stranded DNA molecule;
primer 14 is SEQ ID NO:14, a single-stranded DNA molecule;
primer 15 is SEQ ID NO:15, a single-stranded DNA molecule;
primer 16 is SEQ ID NO: 16;
primer 17 is SEQ ID NO: 17;
primer 18 is SEQ ID NO:18, a single-stranded DNA molecule;
primer 19 is SEQ ID NO: 19;
primer 20 is SEQ ID NO:20, a single-stranded DNA molecule;
primer 21 is SEQ ID NO:21, a single-stranded DNA molecule;
primer 22 is SEQ ID NO: 22;
primer 23 is SEQ ID NO: 23;
primer 24 is SEQ ID NO: 24;
primer 25 is SEQ ID NO: 25;
primer 26 is SEQ ID NO: 26;
primer 27 is SEQ ID NO:27, a single-stranded DNA molecule;
primer 28 is SEQ ID NO: 28;
primer 29 is SEQ ID NO: 29;
primer 30 is SEQ ID NO: 30;
primer 31 is SEQ ID NO: 31;
primer 32 is SEQ ID NO:32, a single-stranded DNA molecule;
primer 33 is SEQ ID NO: 33;
primer 34 is SEQ ID NO:34, a single-stranded DNA molecule;
primer 35 is SEQ ID NO: 35;
primer 36 is SEQ ID NO: 36;
primer 37 is SEQ ID NO: 37;
primer 38 is SEQ ID NO: 38;
primer 39 is SEQ ID NO: 39;
primer 40 is SEQ ID NO: 40;
primer 41 is SEQ ID NO: 41;
primer 42 is SEQ ID NO: 42;
primer 43 is SEQ ID NO: 43;
primer 44 is SEQ ID NO:44, a single-stranded DNA molecule;
primer 45 is SEQ ID NO:45, a single-stranded DNA molecule;
primer 46 is SEQ ID NO: 46;
primer 47 is SEQ ID NO:47, a single-stranded DNA molecule;
primer 48 is SEQ ID NO:48, a single-stranded DNA molecule;
primer 49 is SEQ ID NO:49, or a single-stranded DNA molecule thereof;
primer 50 is SEQ ID NO: 50;
primer 51 is SEQ ID NO: 51;
primer 52 is SEQ ID NO: 52;
primer 53 is SEQ ID NO:53, a single-stranded DNA molecule;
primer 54 is SEQ ID NO:54, or a single-stranded DNA molecule thereof;
primer 55 is SEQ ID NO: 55;
primer 56 is SEQ ID NO: 56;
primer 57 is SEQ ID NO: a single-stranded DNA molecule as set forth in 57;
primer 58 is SEQ ID NO: 58;
primer 59 is SEQ ID NO: 59;
primer 60 is SEQ ID NO: 60;
primer 61 is SEQ ID NO: 61;
primer 62 is SEQ ID NO: 62;
primer 63 is SEQ ID NO: 63;
primer 64 is SEQ ID NO: 64;
primer 65 is SEQ ID NO: 65;
primer 66 is SEQ ID NO: 66;
primer 67 is SEQ ID NO:67, a single stranded DNA molecule;
primer 68 is SEQ ID NO: 68;
primer 69 is SEQ ID NO: 69;
primer 70 is SEQ ID NO: 70;
primer 71 is SEQ ID NO: 71;
primer 72 is SEQ ID NO: 72;
primer 73 is SEQ ID NO: 73;
primer 74 is SEQ ID NO:74, or a single-stranded DNA molecule as shown.
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, primer 52, primer 53, primer 54, primer 55, primer 56, primer 57, primer 58, primer 59, primer 60, primer 61, primer 62, primer 63, primer 64, primer 65, primer 66, primer 67, primer 68, primer 69, primer 70, primer 71, primer 72, primer 74 and primer 150 in a molar ratio: 150:70:70:140:140:180:180:170:170:300:300:160:160:130:130:130:130:120:120:80:80:90:90:140:140:180:180:100:100:150:150:180:180:210:210:120:120:200:200:210:210:160:160:400:400:180:180:120:120:120:120:200:200:200:200:250:250:400:400:150:150:230:230:130:130:190:190:210:210:140:140:300:300.
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, primer 51, primer 53, primer 55, primer 57, primer 59, primer 61, primer 63, primer 65, primer 67, primer 69, primer 71 and primer 73 are all fluorescently labeled.
4. The primer combination of claim 3, wherein:
primer 1, primer 3, primer 5, primer 7, primer 9, primer 11, primer 13, primer 15 and primer 17 were labeled with FAM;
primer 19, primer 21, primer 23, primer 25, primer 27, primer 29 and primer 31 are labeled with HEX;
primer 33, primer 35, primer 37, primer 39, primer 41, primer 43 and primer 45 are labeled with TAMRA;
primer 47, primer 49, primer 51, primer 53, primer 55, primer 57 and primer 59 are labeled with ROX;
primer 61, primer 63, primer 65, primer 67, primer 69, primer 71 and primer 73 are labeled with SID.
5. A multiplex amplification system based on a Y-STR locus and a Y-Indels locus, comprising a primer combination according to any one of claims 1 to 4;
the Y-STR locus comprises DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, Y _ GATA _ H4, DYS438, DYS439, DYS460, DYS533, DYS576, DYS449, DYS518, dysf 387S1, DYS627, DYS570, DYS447, DYS444, DYS549, DYS557, DYS643, DYS481, DYS596, DYS527a/b, DYS508, DYS593, and DYS645;
the Y-Indels loci include rs771783753, rs199815934, and rs759551978.
6. The multiplex amplification system of claim 5, wherein:
the concentration of the primer 1, the primer 2, the primer 31, the primer 32, the primer 61 and the primer 62 in the composite amplification system is 150mM;
the concentration of the primer 3 and the primer 4 in the composite amplification system is 70mM;
the concentration of the primer 5, the primer 6, the primer 25, the primer 26, the primer 71 and the primer 72 in the composite amplification system is 140mM;
the concentration of primer 7, primer 8, primer 27, primer 28, primer 33, primer 34, primer 47 and primer 48 in the multiplex amplification system was 180mM;
the concentration of the primer 9 and the primer 10 in the composite amplification system is 170mM;
the concentration of the primer 11, the primer 12, the primer 73 and the primer 74 in the composite amplification system is 300mM;
the concentration of primer 13, primer 14, primer 43 and primer 44 in the multiplex amplification system is 160mM;
the concentration of the primer 15, the primer 16, the primer 17, the primer 18, the primer 65 and the primer 66 in the composite amplification system is 130mM;
the concentration of primer 19, primer 20, primer 37, primer 38, primer 49, primer 50, primer 51 and primer 52 in the multiplex amplification system is 120mM;
the concentration of the primer 21 and the primer 22 in the composite amplification system is 80mM;
the concentration of the primer 23 and the primer 24 in the composite amplification system is 90mM;
the concentration of the primer 29 and the primer 30 in the composite amplification system is 100mM;
the concentration of the primer 35, the primer 36, the primer 41, the primer 42, the primer 69 and the primer 70 in the composite amplification system is 210mM;
the concentration of the primer 39, the primer 40, the primer 53, the primer 54, the primer 55 and the primer 56 in the composite amplification system is 200mM;
the concentration of the primer 45, the primer 46, the primer 59 and the primer 60 in the composite amplification system is 400mM;
the concentration of primer 57 and primer 58 in the multiplex amplification system was 250mM;
the concentration of the primer 63 and the primer 64 in the composite amplification system is 230mM;
the concentration of the primer 67 and the primer 68 in the multiplex amplification system was 190mM.
7. 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 to perform the PCR amplification reaction do not include primers required for the PCR amplification reaction.
8. A kit comprising the primer combination of any one of claims 1 to 4, wherein: the kit is used for STR typing of male individuals; the use is for the diagnosis and treatment of non-diseases.
9. A method for preparing the multiplex amplification system according to any one of claims 5 to 7 or the kit according to claim 8, comprising the step of packaging each primer in the primer combination according to any one of claims 1 to 4 separately.
10. Use of a primer combination according to any one of claims 1 to 4 or a multiplex amplification system according to any one of claims 5 to 7 in the preparation of a kit for STR typing in male individuals.
11. Use of the primer combination according to any one of claims 1 to 4 or the multiplex amplification system according to any one of claims 5 to 7 for STR typing of male individuals; the use is for the diagnosis and treatment of non-diseases.
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| CN112176068B (en) * | 2019-07-02 | 2022-08-02 | 公安部物证鉴定中心 | Composite amplification system based on 29Y-STR loci and primer combination used by same |
| CN112695100A (en) * | 2021-01-12 | 2021-04-23 | 郑州高新生物技术有限公司 | STR and SNP genetic marker combined detection system and detection method based on NGS |
| CN113584179A (en) * | 2021-04-14 | 2021-11-02 | 南方医科大学 | Six-color fluorescence labeling detection system for degrading 64 loci on human autosome and sex chromosome of test material typing |
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| WO2014039092A1 (en) * | 2012-09-06 | 2014-03-13 | Life Technologies Corporation | Multiplex y-str analysis |
| CN108531610B (en) * | 2018-02-27 | 2021-08-31 | 苏州阅微基因技术有限公司 | Fluorescence multiplex amplification system of 36Y chromosome STR loci and Y-Indel, kit and application thereof |
| CN109468384B (en) * | 2018-11-15 | 2021-12-28 | 江苏苏博生物医学科技南京有限公司 | Composite amplification detection kit for simultaneously detecting 45Y loci |
| CN109439765B (en) * | 2018-11-20 | 2021-12-21 | 江苏苏博生物医学科技南京有限公司 | Composite amplification detection kit for simultaneously detecting 60 autosomal and Y chromosome loci |
| CN109880912B (en) * | 2019-03-07 | 2022-08-09 | 基点认知技术(北京)有限公司 | Composite amplification kit for 44 human Y chromosome loci and application thereof |
| CN109880913B (en) * | 2019-03-07 | 2022-08-19 | 基点认知技术(北京)有限公司 | Composite amplification kit for 38 human Y chromosome loci and application thereof |
| CN110305967A (en) * | 2019-07-04 | 2019-10-08 | 武汉瑞博科创生物技术有限公司 | The composite amplification reagent kit of 36 Y chromosome str locus seats is detected simultaneously |
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