CN109750110B - Composite amplification kit for 47 human autosomal and Y chromosome loci and application thereof - Google Patents
Composite amplification kit for 47 human autosomal and Y chromosome loci and application thereof Download PDFInfo
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Abstract
The invention relates to a composite amplification kit for 47 human autosomal and Y chromosome loci and application thereof. The invention provides a composite amplification system of 47 loci of human autosome and Y chromosome, which comprises specific primers for amplifying 47 loci, wherein the 47 loci comprise 19 autosome STR loci, 27Y chromosome STR loci and 1 individual identification locus. The 47 pairs of specific primers are subjected to grouped fluorescent labeling by utilizing a six-color fluorescent labeling technology, and the simultaneous efficient, specific and sensitive amplification of the gene loci of the 47 human autosomes and the Y chromosome is realized through the design and optimization of primer sequences and working concentrations. The detection result of the composite amplification system has high individual recognition capability and good data compatibility, and further can be practically used for paternity test and individual recognition, so that the detection cost of human DNA typing is effectively reduced, and the detection working efficiency is improved.
Description
Technical Field
The invention relates to the technical field of molecular genetics and biological detection, in particular to a composite amplification kit for 47 human autosomal and Y chromosome loci and application thereof.
Background
Short Tandem Repeat loci (STRs) are currently a commonly used genetic marker. It is widely existed in prokaryotic and eukaryotic genomes, and is a nucleotide repetitive sequence which is composed of 2-6 nucleotides as repetitive units and is more than dozens to one hundred. Different numbers of core sequences are arranged in tandem repeats, and their lengths exhibit genetic polymorphisms. According to the conservative sequences at two ends, a primer is designed, PCR is carried out, and then polymorphism of STR genetic markers can be detected through polyacrylamide, agarose gel electrophoresis or capillary electrophoresis and the like. Compared with other genetic markers, the STR marker has small STR locus fragments, is easy to amplify, is more suitable for trace and degradation detection materials, and can perform composite amplification due to the similar amplification conditions of all loci, so that the STR marker has the advantages of rapidness, high efficiency, accuracy, sensitivity, large information content and the like. Particularly, in the aspect of establishing a DNA database, compared with the conventional technologies such as RFLP, the STR multiplex amplification technology has great superiority.
With the development of domestic STR human identity recognition kits and DNA database library construction projects, a kit product capable of simultaneously obtaining two sets of STR locus typing data of human autosomal chromosomes and Y chromosomes in one amplification detection can be realized, and is more and more popular with customers. With the expansion of database construction market, a kit with higher individual identification rate, more detection loci and better compatibility is urgently needed. The STR kit of the autosomal chromosomes and the Y chromosomes by utilizing the five-color fluorescence detection technology cannot meet the requirements of the current market, and the establishment, the application and the improvement of the six-color fluorescence detection technology can better meet the requirements of the STR locus fluorescence detection kit of the autosomal chromosomes and the Y chromosomes.
With the wider application of STR detection kits, rapid amplification is also an important performance for improving the performance of the kits, and the improvement of the amplification speed greatly reduces the waste of time cost and improves the working efficiency. The amplification time of the traditional kit is about 3-4 hours, and the amplification time of the golden eye20A kit developed earlier by the applicant is about 2.5 hours. Therefore, it is still necessary to further shorten the amplification time and improve the performance and working efficiency of the kit.
In view of the above, there is a need in the art of DNA identification to develop a locus amplification system that can achieve one reaction while amplifying more loci, provide more information, have better compatibility, and have faster amplification speed.
Disclosure of Invention
In order to solve the technical problems of the prior art that the quantity of STR loci detected by multiplex amplification at the same time is limited, the compatibility of a kit is poor and the like, the invention provides a multiplex amplification kit capable of detecting 47 human autosomal and Y chromosome loci at the same time and application thereof.
In one aspect, the present invention provides a locus molecular marker for human chromosomal DNA typing, wherein the molecular marker comprises 47 loci as follows: 19 autosomal STR loci: D7S820, TH01, D6S1043, TPOX, penta E, D16S539, CSF1PO, D19S433, FGA, penta D, D8S1179, D18S51, D3S1358, vWA, D5S818, D13S317, D21S11, D2S1338, D12S391; 27Y chromosome STR loci: DYS392, DYS389I, DYS456, DYS389II, DYS19, DYS460, DYS518, DYS437, DYS576, DYS439, Y _ GATA _ H4, DYS533, DYS391, DYS627, DYS570, DYS635, DYS481, DYS393, DYS390, DYS448, DYS438, DYS458, dysf 387S1, DYS385, DYS449;1 personal recognition locus Amelogenin.
In another aspect, the invention provides a composite amplification kit of 47 loci of human autosomal chromosomes and Y chromosomes, which comprises the amplification primers of 47 loci.
It will be understood by those skilled in the art that the products for multiplex amplification of the 47 loci can be prepared in different forms as required, including but not limited to detection reagents, kits, detection or amplification systems, and all products that include amplification primers for the 47 loci and are capable of performing multiplex amplification of the 47 loci are within the scope of the present invention.
Preferably, the primer component of the composite amplification kit is a primer mixture consisting of the amplification primers of the 47 loci.
Preferably, the sequence of the amplification primer is shown as SEQ ID NO. 1-94: wherein, the sequences of the 19 pairs of amplification primers of the autosomal STR loci are as follows: D7S820 and SEQ ID NO. 1-2; TH01, SEQ ID NO. 3-4; D6S1043 and SEQ ID NO. 5-6; TPOX, SEQ ID NO. 7-8; penta E and SEQ ID NO. 9-10; D16S539 and SEQ ID NO. 11-12; CSF1PO, SEQ ID NO. 13-14; D19S433, SEQ ID NO. 15-16; FGA, SEQ ID NO. 17-18; penta D and SEQ ID NO. 19-20; D8S1179 and SEQ ID NO. 21-22; D18S51 and SEQ ID NO. 23-24; D3S1358 and SEQ ID NO. 25-26; vWA, SEQ ID NO. 27-28; D5S818 and SEQ ID NO. 29-30; D13S317 and SEQ ID NO. 31-32; D21S11 and SEQ ID NO. 33-34; D2S1338 and SEQ ID NO. 35-36; D12S391 and SEQ ID NO. 37-38; the sequences of the 27 pairs of amplification primers for the Y chromosome SYR locus were as follows: DYS392 and SEQ ID NO. 39-40; DYS389I and SEQ ID NO. 41-42; DYS456 and SEQ ID NO. 43-44; DYS389II and SEQ ID NO. 45-46; DYS19 and SEQ ID NO. 47-48; DYS460 and SEQ ID NO. 49-50; DYS518 and SEQ ID NO. 51-52; DYS437, SEQ ID NO. 53-54; DYS576 and SEQ ID NO. 55-56; DYS439 and SEQ ID NO. 57-58; y _ GATA _ H4 and SEQ ID NO. 59-60; DYS533, SEQ ID NO. 61-62; DYS391 and SEQ ID NO. 63-64; DYS627 and SEQ ID NO. 65-66; DYS570, SEQ ID NO. 67-68; DYS635 and SEQ ID NO. 69-70; DYS481 and SEQ ID NO. 71-72; DYS393 and SEQ ID NO. 73-74; DYS390 and SEQ ID NO. 75-76; DYS448 and SEQ ID NO. 77-78; DYS438 and SEQ ID NO. 79-80; DYS458 and SEQ ID NO. 81-82; DYF387S1a and SEQ ID NO. 83-84; DYF387S1b and SEQ ID NO. 85-86; DYS385a and SEQ ID NO. 87-88; DYS385b and SEQ ID NO. 89-90; DYS449, SEQ ID NO. 91-92; 1 amplification primer sequences for the sex recognition loci as follows: amelogenin and SEQ ID NO. 93-94.
More preferably, the working concentration of the primers in the multiplex amplification kit during amplification is as follows: 19 working concentrations for autosomal STR loci: D7S 820. Mu.M, TH01 0.1. Mu.M, D6S10430.12. Mu.M, TPOX 0.09. Mu.M, penta E0.13. Mu.M, D16S539 0.11. Mu.M, CSF1PO 0.13. Mu.M, D19S433 0.18. Mu.M, FGA 0.09. Mu.M, penta D0.15. Mu.M, D8S 1179.2. Mu.M, D18S510.03. Mu.M, D3S1358 0.04. Mu.M, WA 0.05. Mu.M, D5S 818. Mu.M, D13S317 0.06. Mu.M, D21S11 0.09. Mu.M, D2S 1338.13. Mu.M, D12S391 0.16. Mu.M. Working concentration of 27 pairs of Y chromosome STR loci: DYS 392.0.04. Mu.M, DYS389 I.0.03. Mu.M, DYS 456.0.05. Mu.M, DYS389 II.0.06. Mu.M, DYS 19.05. Mu.M, DYS 460.06. Mu.M, DYS 518.0.07. Mu.M, DYS 4370.03. Mu.M, DYS 576.04. Mu.M, DYS439 0.04. Mu.M, Y _ GATA _ H4.05. Mu.M, DYS 30.06. Mu.M, DYS391 0.05. Mu.M, DYS 627.0.07. Mu.M DYS570 0.05. Mu.M, DYS635 0.04. Mu.M, DYS481 0.05. Mu.M, DYS393 0.07. Mu.M, DYS390 0.06. Mu.M, DYS448 0.08. Mu.M, DYS 4380.04. Mu.M, DYS458 0.05. Mu.M, DYF387S1a 0.06. Mu.M, DYF387S1b 0.06. Mu.M, DYS385a 0.05. Mu.M, DYS385b 0.05. Mu.M, DYS449 0.06. Mu.M. 1 working concentration for sex recognition loci: amelogenin 0.03. Mu.M.
To achieve simultaneous rapid amplification of the 47 loci, a six-color fluorescent labeling system can be selected for fluorescent dye labeling of the amplification primers.
The specific fluorescent dye labeling method is as follows: dividing the amplification primers of 47 loci into the following five groups, respectively labeling the amplification primers of the five groups by using fluorescent dyes of five different colors, labeling the amplification primers of each group by using the fluorescent dye of the same color, and labeling one amplification primer of each locus by using the fluorescent dye: a first group, DYS392, DYS389I, DYS456, DYS389II, DYS19, DYS460, DYS518, D7S820, TH01, D6S1043; a second group, DYS437, DYS576, DYS439, Y _ GATA _ H4, DYS533, DYS391, DYS627, TPOX, penta E; a third group, amelogenin, DYS570, DYS635, DYS481, DYS393, DYS390, DYS448, D16S539, CSF1PO, D19S433; a fourth group, DYS438, DYS458, DYF387S1, DYS385, DYS449, FGA, penta D; the fifth group, D8S1179, D18S51, D3S1358, vWA, D5S818, D13S317, D21S11, D2S1338, D12S391.
Preferably, the five fluorescent dye markers with different colors are blue, green, yellow, red and purple fluorescent markers respectively; more preferably, wherein the blue label is a 5-FAM or 6-FAM fluorescein molecule, the green label is a HEX, VIC or JOE fluorescein molecule, the yellow is a TAMRA or NED fluorescein molecule, the Red is a ROX or Texas Red-X fluorescein molecule, and the purple is a NH618 fluorescein molecule.
The selection of the fluorescein molecule includes, but is not limited to, the specific fluorescein molecule described above, and one skilled in the art can select other fluorescein molecules having a similar spectrum to the fluorescein molecule described above as desired.
More preferably, the fluorescent dye label is labeled at the 5' end of the primer.
The amplification reaction program of the composite amplification kit is as follows: 1min to 5min at the temperature of 95 ℃ to 98 ℃; 26-35 cycles of 94-98 ℃ for 5-30 s, 58-62 ℃ for 30 s-1 min and 72 ℃ for 20 s-1 min; final extension at 60 deg.c for 5-30 min.
Preferably, the amplification reaction procedure is as follows: 2min at 95 ℃; 30 cycles of 94 ℃ for 5s, 60 ℃ for 45s and 72 ℃ for 45 s; final extension at 60 ℃ for 20min.
When the composite amplification kit for 47 loci is used for amplification, 25 mul of amplification reaction system is as follows: 2.5 times of reaction premix solution 10 mul, 5 times of primer mixture 5 mul, deionized water 8 mul, and template DNA 2 mul, wherein the primer mixture is the amplification primer mixture of 47 loci.
Preferably, the multiplex amplification kit provided by the invention further comprises a reaction premix containing magnesium ions, dNTPs and DNA polymerase, an allele ladder of 47 loci, a DNA standard and a fluorescent molecular weight internal standard.
It will be understood by those skilled in the art that the major components of Polymerase Chain Reaction (PCR) amplification include templates, primers, dNTPs, DNA polymerase, and DNA polymerase buffer, among others.
The DNA polymerase can be an antibody blocking modified or chemically modified hot-start DNA polymerase. Each amplification system (25. Mu.l) typically requires 2U to 4U of Taq DNA polymerase.
The reaction premix comprises dNTP, DNA polymerase and buffer solution of the DNA polymerase; the buffer solution of the DNA polymerase may include: 50mM KCl,10mM Tris HCl (pH 8.3, 25 ℃ C.), 2.0mM MgCl 2 0.1mg/ml BSA (bovine serum albumin), etc. The skilled person can select separate reagents of dNTP, DNA polymerase and buffer matched with the dNTP and DNA polymerase according to actual needs, or select conventional reagent materials for PCR amplification using dNTP, DNA polymerase and buffer of DNA polymerase as a premixed solution, and the like, and the above selection is within the protection scope of the present invention.
Further, the invention also provides a composite amplification method of 47 loci of human autosome and Y chromosome, which is characterized by comprising the following steps:
(1) Sample treatment: extracting the genome DNA of the sample as an amplification template or directly adopting an extraction-free sample as the amplification template;
(2) Amplifying the sample genome DNA obtained in the step (1) by using an amplification primer with a sequence shown in SEQ ID NO. 1-94;
(3) Detecting the fluorescent signal of the amplification product;
(4) Fluorescence signal data were collected and analyzed to obtain DNA typing results for 47 loci.
Wherein the sample comprises one or more of blood, blood stain, semen stain, bone, hair, saliva stain, sweat, and amniotic fluid containing fetal cells; or human blood or oral cavity cells collected by using any carrier of blood card, cotton swab and gauze.
Preferably, the genomic DNA of the extracted sample may be extracted by any one of a Chelex method, a magnetic bead extraction method and an organic extraction method.
Preferably, the amount of the DNA template in the sample is preferably 0.5ng to 4ng.
The amplification reaction can be performed on various reaction thermal cyclers, such as ABI 9700, ABI 9600, ABI2720, bio Rad iCycler, bio Rad Cl000, and the like.
The amplification product is provided with a fluorescent dye label, the fluorescent label can emit a luminescent signal under laser excitation, and the fluorescent signal detection in the step (3) can be carried out electrophoresis and detection by a sequencer or a genetic analyzer and other instruments; wherein, the analysis sequencer includes but is not limited to ABI 377, 310DNA sequencers, and the genetic analyzer includes but is not limited to ABI3100, 3130, 3500, 3730 series genetic analyzer.
Specifically, when detection is performed on a sequencer or a genetic analyzer, an amplification product is mixed with a molecular weight internal standard (marker) and formamide according to a certain proportion, and the mixture enters an instrument capillary or gel for electrophoretic separation. The molecular weight internal standard is composed of a plurality of fluorescence labeling DNA segments with known lengths and is used for calculating the length of the PCR amplification product segment, thereby being capable of judging the gene typing and comparing with allele step.
The collected data such as fluorescence signals can be analyzed on data analysis software such as GeneMapper, geneMarker and GeneScan, and finally STR genotyping maps and data are obtained.
In addition, the invention also provides an amplification primer group, which comprises 47 pairs of primers with sequences shown as SEQ ID NO. 1-94.
Further, the present invention provides the use of said molecular marker or said composite amplification kit or said amplification primer set for individual identification or paternity testing or for the construction of databases of human chromosomal DNA loci.
The design concept of the main technical scheme of the invention is as follows:
1. screening of 47 combinations of human autosomal and Y chromosomal loci
The factors for selecting the target gene locus mainly include genetic polymorphism, compatibility with the existing detection kit, contribution of the newly added gene locus to improvement of individual recognition capability and the like. The inventors analyzed genetic polymorphisms of chromosomes of a total of 52 loci for different individuals, and finally determined the following combinations of 47 chromosomal loci on the basis of sufficient analysis of loci used in existing kits: 19 autosomal STR loci: D7S820, TH01, D6S1043, TPOX, penta E, D16S539, CSF1PO, D19S433, FGA, penta D, D8S1179, D18S51, D3S1358, vWA, D5S818, D13S317, D21S11, D2S1338, D12S391; 27Y chromosome STR loci: DYS392, DYS389I, DYS456, DYS389II, DYS19, DYS460, DYS518, DYS437, DYS576, DYS439, Y _ GATA _ H4, DYS533, DYS391, DYS627, DYS570, DYS635, DYS481, DYS393, DYS390, DYS448, DYS438, DYS458, dysf 387S1, DYS385, DYS449;1 personal recognition locus Amelogenin. On the basis of a market commercialized kit AGCUEX16+19Y kit, the kit provided by the invention is added with 14 highly genetic polymorphic loci such as D12S391, penta D, penta E, D6S1043, DYF387S1, DYS449, DYS460, DYS381, DYS518, DYS533, DYS570, DYS576 and DYS 627. Therefore, the kit of the present invention breaks through the maximum multiplex amplification that can be achieved at present while detecting the number of loci (35), and has significantly improved individual recognition ability and non-paternal discharge rate. The arrangement positions of the 47 loci are shown in FIG. 1.
2. Development of six-color fluorescent labeling technology
The traditional five-color fluorescence labeling technology limits the number of complex amplification gene loci, and several currently commonly used six-color fluorescence technologies have the problem that a genetic analyzer cannot completely and automatically remove mutual permeation among fluorescent dye signals due to too close emission wavelengths of partial fluorescent dyes, so that a permeation peak is generated. There is also a problem that the fluorescence efficiency is low because the maximum absorption wavelength of a fluorescein molecule is far from the excitation wavelength of a genetic analyzer. The physical and chemical properties of fluorescein, the wavelength of excitation light of a detection instrument and other factors are comprehensively considered, and through a large number of screening and comparison experiments, fluorescein with proper wavelength and high energy conversion efficiency is finally selected to be matched with the traditional five-color fluorescence technology for use, so that the six-color fluorescence labeling technology for composite amplification is developed.
Firstly, the optimal amplification fragment length and other factors are comprehensively considered, 47 gene loci are divided into five groups, each group is respectively marked by different fluorescein, amplification products of each gene locus in each group are separated according to the length difference, and two gene loci cannot be overlapped. By reasonably matching the PCR amplification efficiency of the gene locus and the fluorescence efficiency of the fluorescein marker, the amplified fragment has a similar RFU value during capillary electrophoresis analysis, and the balance is obviously improved.
The invention obtains the following preferred groups through a large number of optimization experiments: a first group, DYS392, DYS389I, DYS456, DYS389II, DYS19, DYS460, DYS518, D7S820, TH01, D6S1043; a second group, DYS437, DYS576, DYS439, Y _ GATA _ H4, DYS533, DYS391, DYS627, TPOX, penta E; a third group, amelogenin, DYS570, DYS635, DYS481, DYS393, DYS390, DYS448, D16S539, CSF1PO, D19S433; a fourth group, DYS438, DYS458, DYF387S1, DYS385, DYS449, FGA, penta D; the fifth group, D8S1179, D18S51, D3S1358, vWA, D5S818, D13S317, D21S11, D2S1338, D12S391.
Then, specific primers were designed to flank the repetitive sequences of the above 47 loci, respectively. The basic principle of primer design is as follows: each primer anneals at a temperature near or above 60 ℃. The secondary structures such as primer dimer, hairpin structure inside the primer and the like and cross reaction can not be generated, and the length of the amplified product is between 65 and 500 bp. Amplification tests were performed and optimized for each pair of primers until a clear single amplified band was obtained.
Multiplex amplification assays are performed using primer pairs for each set of loci. After determining that the group has no non-specific amplification phenomenon, no cross reaction and the like, continuously adjusting the concentration of each pair of primers to ensure that the peak value balance of each segment in the group reaches more than 50 percent.
Primers of five groups of loci are respectively marked by blue, green, yellow, red and purple fluorescein. Only one strand of each primer pair is labeled, with the label being at the 5' end of the primer. The blue marker can be 5-FAM, 6-FAM or fluorescein molecules with similar spectra, the green marker can be HEX, JOE, VIC or fluorescein molecules with similar spectra, the yellow marker can be TAMRA, NED or fluorescein molecules with similar spectra, the Red marker can be ROX, texas Red-X or fluorescein molecules with similar spectra, and the purple marker can be NH618 or fluorescein molecules with similar spectra.
Finally, carrying out composite amplification on the five groups of 47 loci, modifying and replacing primers for generating non-specific amplification, adjusting the primer concentration of each locus according to the peak height condition of a product, and determining the optimal working concentration of 47 pairs of primers in a primer mixture through massive optimization and screening so that the integral peak value balance of each locus reaches more than 30%.
The invention has the beneficial effects that:
(1) The invention establishes a composite amplification system for simultaneously amplifying 19 autosomal STR loci, 27Y chromosome STR loci and 1 Amelogenin locus for the first time, realizes the simultaneous high-efficiency, specific and sensitive amplification of 47 autosomal and Y chromosome loci by ingenious primer sequences, concentration design and improved six-color fluorescence labeling technology, and is a composite amplification system with the largest number of loci capable of realizing the simultaneous amplification by the currently reported human DNA typing technology;
(2) The 47 combinations of human autosomal chromosomes and Y chromosome loci provided by the invention are added with a plurality of loci with high genetic polymorphism on the basis of all loci used by DNA typing detection reagents at home and abroad, so that the individual identification capability of detection is obviously improved;
(3) The combination of 47 human autosomes and Y chromosome loci provided by the invention integrates all loci adopted by the current DNA typing detection reagent at home and abroad, has good analysis data compatibility, can be compatible with the existing autosomes and Y chromosome data in the DNA database at present in China, has high compatibility with new generation products, and overcomes the problem of data compatibility of the detection reagent in the prior art;
(4) The genetic information of the 47 human autosomes and the Y chromosome loci provided by the invention is higher than the information amount obtained by simultaneously using 2-3 similar products in the prior art, so that the labor, time and material cost are saved by more than 50% no matter in the links of PCR amplification and genetic analyzer detection, and the detection working efficiency is improved;
(5) The composite amplification kit provided by the invention has strong material detection adaptability, and one kit can amplify samples of various detection materials of human blood or oral cells, which are collected by any carrier such as filter paper, FTA card, cotton swab, gauze and the like, and human genome DNA extracted by any method of a Chelex method, a magnetic bead extraction method or an organic extraction method;
(6) The composite amplification system provided by the invention has stronger amplification specificity and wider temperature tolerance range, and can ensure that different PCR amplification instruments can obtain better amplification results.
(7) The composite amplification system provided by the invention can be widely applied to the construction of an autosomal DNA database and a Y STR haplotype database, and is also suitable for field case detection, particularly the detection of male components in male and female mixed spots, case investigation and family genetic search.
Drawings
FIG. 1 is a schematic diagram of the arrangement of 47 human autosomal and Y chromosome loci according to the present invention.
FIG. 2 is an Allelic Ladder map, which is an Allelic typing standard, of the multiplex amplification kit provided by the present invention.
FIG. 3 is a typing map of the 47 loci of 9948DNA in the multiplex amplification kit provided by the present invention.
FIG. 4 is a typing map of the 9948DNA of comparative example 1 at 47 loci.
FIG. 5 is a typing map of the 9948DNA of comparative example 2 at 47 loci.
FIG. 6 is a typing map of 47 loci of the suspected father identified by the genetic relationship in example 4.
FIG. 7 is a typing map of 47 loci of suspected son identified by genetic relationship in example 4.
FIG. 8 is a typing map of 47 loci of sample 1 in the database construction in example 5.
FIG. 9 is a typing map of 47 loci of sample 2 in the database construction in example 5.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 development of a kit for multiplex amplification of 47 human autosomal and Y chromosomal loci
1. Design of 47 human autosomal and Y chromosome locus composite amplification primers
For a multiplex amplification system, especially for the multiplex amplification system of 47 loci in the invention, the requirements on the specificity of a primer sequence, a secondary structure, the stability of combination with a target sequence, amplification efficiency and the like are extremely high, and 47 pairs of primers for specifically and efficiently amplifying 47 loci are obtained through continuous cyclic experiments of amplification, optimization and amplification, wherein the specific sequence list of the primers is shown in the sequence table 1:
TABLE 1 amplification primers for 47 loci
Carrying out fluorescent labeling on the primers with different colors according to the following grouping modes of corresponding loci: a first group, DYS392, DYS389I, DYS456, DYS389II, DYS19, DYS460, DYS518, D7S820, TH01, D6S1043; a second group, DYS437, DYS576, DYS439, Y _ GATA _ H4, DYS533, DYS391, DYS627, TPOX, penta E; a third group, amelogenin, DYS570, DYS635, DYS481, DYS393, DYS390, DYS448, D16S539, CSF1PO, D19S433; a fourth group, DYS438, DYS458, DYF387S1, DYS385, DYS449, FGA, penta D; the fifth group, D8S1179, D18S51, D3S1358, vWA, D5S818, D13S317, D21S11, D2S1338, D12S391. Groups 1-5 were labeled with blue, green, yellow, red and violet fluorescein, respectively. Only one strand of each primer pair is labeled, with the label at the 5' end of the primer. Wherein the blue marker is selected from 6-FAM, the green marker is selected from HEX, the yellow marker is selected from TAMRA, the red marker is selected from ROX, and the purple marker is selected from NH618.
2. Optimization of primer concentration in multiplex amplification reaction system
For a composite amplification system for amplifying 47 loci simultaneously, in order to realize higher amplification equilibrium degree, the concentration ratio of 47 pairs of primers in the composite amplification system is continuously adjusted, the amplification equilibrium degree is gradually improved, and the optimal primer concentration is finally obtained as follows: D7S 820. Mu.M, TH01 0.1. Mu.M, D6S 1043.12. Mu.M, TPOX 0.09. Mu.M, penta E0.13. Mu.M, D16S539 0.11. Mu.M, CSF1PO 0.13. Mu.M, D19S4330.18. Mu.M, FGA 0.09. Mu.M, penta D0.15. Mu.M, D8S1179 0.2. Mu.M, D18S51 0.03. Mu.M, D3S1358 0.04. Mu.M, vWA 0.05. Mu.M, D5S 818. Mu.M, D13S 317. 0.06. Mu.M, D21S110.09. Mu.M, D2S 1338.13. Mu.M, D12S391 0.16. Mu.M. Working concentration of 27 pairs of Y chromosome STR loci: DYS392 0.04 μ M, DYS389I 0.03 μ M, DYS456 0.05 μ M, DYS389II0.06 μ M, DYS 19.05 μ M, DYS460 0.06 μ M, DYS518 0.07 μ M, DYS4370.03 μ M, DYS576 0.04 μ M, DYS439 0.04 μ M, Y _ GATA _ H4.05 μ M, DYS5330.06 μ M, DYS391 0.05 μ M, DYS627 0.07 μ M, DYS570 0.05 μ M, DYS635 0.04 μ M, DYS4810.05 μ M, DYS393 0.07 μ M, DYS 0.06 μ M, DYS448 0.08 μ M, DYS438 μ M, DYS458 μ M, DYS 458.05 μ M, DYS385 μ M, DYS 06 μ M, DYS385A 1.06 μ M, DYS449 0.05 μ M, DYS 05 μ M, DYS 06 μ M, DYS438 μ M, DYS 06 μ M, DYS 05 μ M, DYS385 μ M, DYS 06 μ M.1 working concentration for sex recognition loci: amelogenin 0.03. Mu.M.
The 47 chromosomal locus multiplex amplification kit consisted of the following components (Table 2).
TABLE 2 composite amplification kit composition
Wherein the primer mixture is a mixture of 47 pairs of primers with sequences shown as SEQ ID NO. 1-94.
EXAMPLE 2 establishment of a Complex amplification System and procedure for 47 chromosomal loci
The optimization of annealing temperature and cycle number was performed for the 47 loci amplification primers, resulting in an optimal reaction program: keeping the temperature at 95 ℃ for 2 minutes; heat preservation at 94 ℃ for 5 seconds, at 60 ℃ for 45 seconds and at 72 ℃ for 45 seconds, and the step is operated for 30 cycles; keeping the temperature at 60 ℃ for 20 minutes; keeping the temperature at 4-10 ℃.
Example 3 genotyping of 9948 cell line Using multiplex amplification kit
The kit provided by the invention is used for carrying out composite amplification on the gene loci of 47 human autosomes and Y chromosomes on a 9948 cell strain. A template DNA derived from 9948 cell line was extracted by the chelex100 method. The amplification reactions were performed on an ABI 9700 thermal cycler, the electrophoresis and detection were performed on an ABI 3130 genetic analyzer, and the data analysis was performed using GeneMapper IDX v1.2 software. The reagent materials used, such as the allelic ladder (ladder), are commercially available and are conventional materials commonly used by those skilled in the art.
1. DNA was extracted by the chelex100 method (for a specific method, refer to Forensic DNA Protocol, human Press, 1998):
(1) Mu.l of the cultured 9948 cell line was taken in a 500. Mu.l centrifuge tube.
(2) The chelex solution was mixed by shaking to suspend the chelex well, and 195. Mu.l of 5% chelex100 (l 00 200mesh, available from Bio Rad) was added to each tube, followed by 5. Mu.l of proteinase K (20 mg/ml, available from Kuh-Chazu Biochemical Co., ltd.).
(3) The sample was shaken and after 2 hours of incubation at 56 ℃ on a constant temperature metal bath, the sample was taken out and shaken for 2 minutes.
(4) Boiling for 8-10 min, and centrifuging at 13000rpm for 3 min.
(5) About 150. Mu.l of the supernatant was carefully aspirated, transferred to a new tube, and 1. Mu.l of the 10. Mu.l PCR reaction was used as a template.
2. Polymerase Chain Reaction (PCR) amplification
(1) Taking the buffer solution and the primer mixture, preparing a mixed solution according to the following table, shaking, uniformly mixing, subpackaging into PCR reaction tubes, adding the template DNA, and preparing into a 25 mu l reaction system (table 3).
TABLE 3 PCR amplification reaction System
Wherein the primer mixture is a mixture of 47 pairs of primers with sequences shown as SEQ ID NO. 1-94; the reaction premix comprises: 50mM KCl,10mM Tris HCl (pH 8.3, 25 ℃ C.), 2.0mM MgCl 2 0.1mg/ml BSA (bovine serum albumin) and 0.2mM each of dNTPs (dNTPs are equimolar mixtures of the four deoxyribonucleotides dATP, dTTP, dCTP, dGTP), and Taq DNA polymerase required for the reaction. The Taq DNA polymerase used is an antibody blocking modified or chemically modified hot-start DNA polymerase, and each amplification system (25 ul) requires 2U-4U of Taq DNA polymerase.
(2) A thermal cycler (ABI 9700 PCR) was set up according to the following reaction conditions, and a PCR reaction tube was placed in the apparatus to start amplification of gene fragments. Keeping the temperature at 95 ℃ for 2 minutes; heat preservation is carried out for 30 cycles at 94 ℃ for 5 seconds, 60 ℃ for 45 seconds and 72 ℃ for 45 seconds; keeping the temperature at 60 ℃ for 20 minutes, and keeping the temperature at 4-10 ℃ until the sample is taken out.
3. After the amplification reaction was completed, the reaction tube was taken out, and electrophoresis and detection were carried out using ABI 3130 genetic analyzer.
(1) A mixture was prepared by mixing (0.5. Mu.l of an internal molecular weight standard + 10. Mu.l of deionized formamide) × (the number of samples).
(2) After mixing, the mixture was dispensed into 10. Mu.l tubes, and then 1. Mu.l of amplification product or allelic ladder (ladder, as shown in FIG. 2) was added, followed by brief centrifugation to collect the liquid at the bottom of the tube of the centrifuge tube.
(3) The sample was denatured at 95 ℃ for 4 minutes and then rapidly cooled on ice for 4 minutes to completely denature the DNA and maintain the denatured state.
(4) The sample is put into a sample tray of a genetic analyzer, instrument parameters (sample introduction voltage of 3kV and sample introduction time of 4 seconds) are set, and electrophoresis detection is started.
(5) After about 50 minutes, the electrophoresis was terminated and the experimental data were analyzed by GeneMapper software to obtain a pattern and typing results.
The results are shown in fig. 3, the sequence information of the 47 human autosomal and Y chromosome loci of the 9948 cell line obtained by using the kit provided by the present invention is consistent with the information in the database, and it is proved that the kit provided by the present invention can realize the accurate detection of the 47 chromosome loci at one time.
Comparative example 1 amplification Using primers of different sequences
Typing of the 47 chromosomal loci of the 9948 cell line was carried out using the composite amplification system and procedure in examples 1 and 2 using the detection method in example 3 by replacing the primers of the DYS460, DYS481, TPOX and D3S1358 loci, respectively, with the primer sequences in the following table, and as a result, as shown in fig. 4, DYS460 appeared in the blue group with a non-specific amplification peak similar to n-10 before its main peak; DYS481 is in yellow group, and has obvious strip peaks with regular intervals after the main peak, and the peak height is gradually reduced; TPOX is in a green group, obvious nonspecific amplification is generated at the position 80bp in front of an analysis area of the green group data, and a main peak is obviously lower; D3S1358 has a more obvious n-1 phenomenon in the purple group. The results show that the primer pairs in table 4 used for replacement may cause adverse effects in the DNA typing process, and are not suitable for the primer combination of the present invention, which indicates that the adjustment of the primer sequence significantly affects the detection efficiency of the kit.
TABLE 4 primer sequences used in comparative example 1
Comparative example 2 Effect of variation in primer concentration
The primer concentrations of the DYS576 and D21S11 loci were replaced with the primer concentrations in Table 5, and the detection method in example 3 was used to type the 47 chromosomal loci of the 9948 cell line using the multiplex amplification system and procedure in examples 1 and 2, and the results are shown in FIG. 5. After the primer concentration of DYS576 is adjusted, the product peak height of the gene locus in an electrophoretogram is obviously changed and is more than 2 times lower than the peak height of adjacent gene loci DYS437 and DYS439, and the balance cannot meet the requirements of the kit; after the primer concentration of the D21S11 is adjusted, the peak height is correspondingly and obviously increased, and the requirement on the equilibrium of the kit can not be met.
TABLE 5 primer concentrations used in comparative example 2
| Genetic loci | Concentration of primer pair |
| DYS576 | 0.04μM |
| D21S11 | 0.09μM |
Example 4 genetic relationship identification Using multiplex amplification kit
The kit provided by the invention is used for carrying out composite amplification of 47 human autosomal and Y chromosome loci on 2 blood card samples from suspected fathers and son. PCR amplification is carried out by adopting a method of punching and directly amplifying blood cards. The amplification reactions were performed on an ABI 9700 thermal cycler, the electrophoresis and detection were performed on an ABI 3130 genetic analyzer, and the data analysis was performed using GeneMapper IDX v1.2 software.
1. Blood samples were each punched out of blood cards from grandfather two using a punch 1.2mm in diameter, noting that the position of the punch was sufficiently penetrated by the blood sample and was completely dried. And placing the punched blood card as a template in a PCR amplification tube for later use.
2. Polymerase Chain Reaction (PCR) amplification
(1) Taking the buffer solution and the primer mixture, preparing a mixed solution according to the table 6, shaking, uniformly mixing, subpackaging into PCR reaction tubes, adding the template DNA, and preparing into a 25 mu l reaction system (table 6).
TABLE 6 PCR amplification reaction System
Wherein the primer mixture is a mixture of 47 pairs of primers with sequences shown as SEQ ID NO. 1-94; the reaction premix comprises: 50mM KCl, l0mM Tris HCl (pH 8.3, 25 ℃ C.), 2.0mM MgCl 2 0.1mg/ml BSA (bovine serum albumin) and 0.2mM each of dNTPs. dNTPs are an equimolar mixture of four deoxyribonucleotides (dATP, dTTP, dCTP, dGTP), and Taq DNA polymerase required for the reaction. The Taq DNA polymerase used is an antibody blocking modified or chemically modified hot-start DNA polymerase, and each amplification system (25 ul) requires 2U-4U of Taq DNA polymerase.
(2) A thermal cycler (ABI 9700 PCR) was set up according to the following reaction conditions, and a PCR reaction tube was placed in the apparatus to start amplification of gene fragments. Keeping the temperature at 95 ℃ for 2 minutes; heat preservation is carried out for 30 cycles at 94 ℃ for 5 seconds, 60 ℃ for 45 seconds and 72 ℃ for 45 seconds; keeping the temperature at 60 ℃ for 20 minutes, and keeping the temperature at 4-10 ℃ until the sample is taken out.
3. After the amplification reaction was completed, the reaction tube was taken out, and electrophoresis and detection were carried out using ABI 3130 genetic analyzer.
(1) A mixture was prepared by mixing (0.5. Mu.l of an internal molecular weight standard + 10. Mu.l of deionized formamide) × (the number of samples).
(2) Mixing, packaging 10 μ l each tube, and adding 1 μ l each amplification product. Another 1. Mu.l of allele trap (ladder, as shown in FIG. 2) was added to the tube containing the above mixture as the amplification product, and the liquid was collected at the bottom of the tube by brief centrifugation.
(3) The sample was denatured at 95 ℃ for 4 minutes and then rapidly cooled on ice for 4 minutes to completely denature the DNA and maintain the denatured state.
(4) The sample is put into a sample tray of a genetic analyzer, instrument parameters (sample introduction voltage 3kV and sample introduction time 4 seconds) are set, and electrophoresis detection is started.
(5) After about 50 minutes, the electrophoresis was terminated and the experimental data were analyzed by GeneMapper software to obtain the pattern and typing results. The results are shown in fig. 6 (suspected father) and fig. 7 (suspected son), the typing information of the loci of 47 human autosomes and Y chromosomes obtained by the suspected father and the suspected son obtained by the kit provided by the invention is compared, and the typing of the loci of 47 human autosomes and Y chromosomes of the suspected father and the suspected son is consistent, so that the genetic rule is met. And by combining the actual situation, the genetic relationship judgment supporting the suspected parent and child as the parent and child can be made.
Example 5 judicial identification Using multiplex amplification kits
The kit provided by the invention is used for carrying out composite amplification on 47 human autosomal and Y chromosome loci on 2 common blood card samples in the market. PCR amplification is carried out by adopting a blood card punching direct amplification method. The amplification reactions were performed on an ABI 9700 thermal cycler, the electrophoresis and detection were performed on an ABI 3130 genetic analyzer, and the data analysis was performed using GeneMapper IDX v1.2 software.
1. A punch with the diameter of 1.2mm is used for punching blood slices on 2 blood card samples respectively, and the positions where the blood slices are punched need to be noticed to fully permeate the blood samples and be completely dried. And placing the punched blood card as a template in a PCR amplification tube for later use.
2. Polymerase Chain Reaction (PCR) amplification
(1) Taking the buffer solution and the primer mixture, preparing a mixed solution according to the table 7, shaking, uniformly mixing, subpackaging into PCR reaction tubes, adding the template DNA, and preparing into a 25 mu l reaction system (table 7).
TABLE 7 PCR amplification reaction System
Wherein the primer mixture is a mixture of 47 pairs of primers with sequences shown as SEQ ID NO. 1-94; the reaction premix comprises: 50mM KCl, l0mM Tris HCl (pH 8.3, 25 ℃ C.), 2.0mM MgCl 2 0.1mg/ml BSA (bovine serum albumin) and 0.2mM each of dNTPs. dNTPs are an equimolar mixture of four deoxyribonucleotides (dATP, dTTP, dCTP, dGTP), and Taq DNA polymerase required for the reaction. The Taq DNA polymerase used is an antibody blocking modified or chemically modified hot-start DNA polymerase, and each amplification system (25 ul) requires 2U-4U of Taq DNA polymerase.
(2) A thermal cycler (ABI 9700 PCR) was set up according to the following reaction conditions, and a PCR reaction tube was placed in the apparatus to start amplification of gene fragments. Keeping the temperature at 95 ℃ for 2 minutes; keeping the temperature at 94 ℃ for 5 seconds, keeping the temperature at 60 ℃ for 45 seconds, keeping the temperature at 72 ℃ for 45 seconds, and running for 30 cycles; keeping the temperature at 60 ℃ for 20 minutes, and keeping the temperature at 4-10 ℃ until the sample is taken out.
3. After the amplification reaction is completed, the reaction tube is taken out, and electrophoresis and detection are performed by using an ABI 3130 genetic analyzer.
(1) A mixture was prepared from (0.5. Mu.l of molecular weight internal standard + 10. Mu.l of deionized formamide) × (number of samples).
(2) Mixing, packaging 10 μ l each tube, and adding 1 μ l each amplification product. Another 1. Mu.l of allele trap (ladder, as shown in FIG. 2) was added to the tube containing the above mixture as the amplification product, and the liquid was collected at the bottom of the tube by brief centrifugation.
(3) The sample was denatured at 95 ℃ for 4 minutes and then rapidly cooled on ice for 4 minutes to completely denature the DNA and maintain the denatured state.
(4) The sample is put into a sample tray of a genetic analyzer, instrument parameters (sample introduction voltage 3kV and sample introduction time 4 seconds) are set, and electrophoresis detection is started.
(5) After about 50 minutes, the electrophoresis was terminated and the experimental data were analyzed by GeneMapper software to obtain a pattern and typing results. The results of the test material 1 and the test material 2 are respectively shown in fig. 8 and fig. 9, and the parting information of 47 autosomal and Y chromosome loci of the autosomal and Y chromosome STR databases obtained by using the kit provided by the invention is used for building samples, and the parting results are respectively exported and recorded into the databases.
It will be appreciated by persons skilled in the art that the foregoing description is only an example of the invention and that the scope of the invention as claimed is not limited solely to the specific embodiments disclosed herein. Any equivalent embodiments are to be considered within the scope of the present invention. Indeed, various modifications and variations of the present invention are possible in light of the above teachings, and it is intended that such modifications and variations be included within the scope of the appended claims.
Sequence listing
<110> Kyoto cognitive technology (Beijing) Ltd
Composite amplification kit for <120> 47 human autosomal and Y chromosome loci and application thereof
<160> 94
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agtgcagtgg tgcgatctc 19
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cacaccagaa ggaataaaaa caggca 26
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgcaatctca tccactattt gtct 24
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tctctttctt ctggagctga gaag 24
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cccccacctt cctctgcttc 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tgagctcagg agatcaagac cagc 24
<210> 10
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggtggtatgg ctcatttatg aagttaat 28
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
actgagaggc tactttctga ccca 24
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ttagcgtttg tgtgtgcatc tgta 24
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tgttccaacc tgagtctgcc a 21
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cttgcacata ctctaggggc a 21
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
tcaagtctag tctgggcaac atagtg 26
<210> 16
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aaaatgctac aagaactaac atgaaact 28
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
actaattgct attaggacat cttaactggc 30
<210> 18
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ttccctctac tcagaaacaa ggacat 26
<210> 19
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ctaattgtac cttgggaggc tgagac 26
<210> 20
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ggttaaatat ctcttcaaat cttttgccca 30
<210> 21
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tttgtatttc atgtgtacat tcgtatct 28
<210> 22
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tatcctgtag attattttca ctgtggtt 28
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgcacttcac tctgagtgac aaat 24
<210> 24
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tggtgtgtgg agatgtctta caat 24
<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gccatattca cttgcccact t 21
<210> 27
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tattattttg tgaactcctc agactgatcc 30
<210> 28
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
agatgataga tacataggat agatagagac 30
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ctggcttacc ccctcatttt g 21
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgctttttag ccaagtgatt ccaa 24
<210> 31
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgtattgcaa gcacttagtt acatttctt 29
<210> 32
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ataacagtct gaaagtacaa gtggggaa 28
<210> 33
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
<210> 34
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
cataaacact gagaagggag aaacactg 28
<210> 35
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
attggggagc tgtttcagaa c 21
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ggactctgca ggagtctatc t 21
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gttcctgtaa ccccagctac tca 23
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ttcttctgcc cttggaagtc aga 23
<210> 39
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
gtgtttgtta tttaaaagcc aagaaggaaa 30
<210> 40
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cctaccaatc ccattcctta gtaaata 27
<210> 41
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ctcatctgta ttatctatgt gtgtgtct 28
<210> 42
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
acagacagac agatacatag ataataca 28
<210> 43
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
acattggact ctaatttttt cagttttg 28
<210> 44
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
atcaactcag cccaaaactt cttaa 25
<210> 45
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ctcatctgta ttatctatgt gtgtgtct 28
<210> 46
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gaggaacaca attatccctg agtagcag 28
<210> 47
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cagtctaggt atgagatcaa attgactg 28
<210> 48
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gatattactt ggactggaag acaaggac 28
<210> 49
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gttctcagct cagaactata gtcctc 26
<210> 50
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tctatctatc tatctaccta tcttctatct 30
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gatctcgtca ttgcactcca gc 22
<210> 52
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tttctgttca agtcttgagt cttgaact 28
<210> 53
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
<210> 54
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tagatagaca tcattcacag atgatagatg 30
<210> 55
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
atcccttggg ctgaggagtt caa 23
<210> 56
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gggagtaata agcgtatttg tcttggct 28
<210> 57
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tgtcctgaat ggtacttcct aggttttctt 30
<210> 58
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
ctggcttgga attcttttac ccatcatctc 30
<210> 59
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
cagagatgtt ggttttctga tacacattga 30
<210> 60
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
aaaattttac atagcccact tgttaaacaa 30
<210> 61
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
agtgattcat gggaaatcat aatgcaca 28
<210> 62
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
aagttgagag atgatgaaag tagatagatg 30
<210> 63
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
<210> 64
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
caagtgtccc ctagggacta agga 24
<210> 65
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
agataatgcc actgcactcc acc 23
<210> 66
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
acatgagtaa ttttgacaat atacactagc 30
<210> 67
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
atcctggctg tgtcctccaa gt 22
<210> 68
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
tgaaatgcag atattcccta tctacaaa 28
<210> 69
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tggaatgctc tcttggcttc tca 23
<210> 70
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
acattgttta tagcagcaaa attcacagtt 30
<210> 71
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
agtgttgcga gagttagatg tctatgt 27
<210> 72
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
aaaaatcaga acacagagcc ccac 24
<210> 73
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
atgtggctgt gagtcaatta aaccctt 27
<210> 74
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gttacaaaaa gaatggccta taatctaact 30
<210> 75
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
tgaggacaga gactccagta ttggt 25
<210> 76
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
atgaaaacat tgcaatgtgt atactcag 28
<210> 77
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
aaggaagata tcaaagagca cccgt 25
<210> 78
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
tcatatttct ggccggtctg gaaatttatc 30
<210> 79
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ccaaaattag tggggaatag ttgaacggta 30
<210> 80
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
gcaacaagag tgaaactcca tttcaaa 27
<210> 81
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
ggaatgaaac tccaatgaaa gaaaga 26
<210> 82
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
<210> 83
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
tgggtgacag agctagattc catt 24
<210> 84
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
agaagtgtga gaagtgctac caca 24
<210> 85
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
tgggtgacag agctagattc catt 24
<210> 86
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
agaagtgtga gaagtgctac caca 24
<210> 87
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
tgaacctgaa atgtaaaggg tgtcatga 28
<210> 88
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
tctatctatt ccaattacat agtcctcc 28
<210> 89
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
tgaacctgaa atgtaaaggg tgtcatga 28
<210> 90
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
tctatctatt ccaattacat agtcctcc 28
<210> 91
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
taggctagag attcttggag tctctc 26
<210> 92
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
<210> 93
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
tccaggtctc caactttaag actat 25
<210> 94
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
aggagacttc ttattttaaa gaggtctaga 30
Claims (5)
1. A composite amplification kit for 47 loci of human autosomal chromosomes and Y chromosome is characterized by comprising the following amplification primers for 47 loci: 19 autosomal STR loci: D7S820, TH01, D6S1043, TPOX, penta E, D16S539, CSF1PO, D19S433, FGA, penta D, D8S1179, D18S51, D3S1358, vWA, D5S818, D13S317, D21S11, D2S1338, D12S391; 27Y chromosome STR loci: DYS392, DYS389I, DYS456, DYS389II, DYS19, DYS460, DYS518, DYS437, DYS576, DYS439, Y _ GATA _ H4, DYS533, DYS391, DYS627, DYS570, DYS635, DYS481, DYS393, DYS390, DYS448, DYS438, DYS458, dysf 387S1, DYS385, DYS449;1, identifying a gene locus Amelogenin individually;
the sequence of the amplification primer is shown as SEQ ID NO. 1-94: wherein, the sequences of the 19 pairs of amplification primers of the autosomal STR locus are as follows: D7S820 and SEQ ID NO. 1-2; TH01, SEQ ID NO. 3-4; D6S1043 and SEQ ID NO. 5-6; TPOX and SEQ ID NO. 7-8; penta E and SEQ ID NO. 9-10; D16S539 and SEQ ID NO. 11-12; CSF1PO, SEQ ID NO. 13-14; D19S433 and SEQ ID NO. 15-16; FGA, SEQ ID NO. 17-18; penta D and SEQ ID NO. 19-20; D8S1179 and SEQ ID NO. 21-22; D18S51 and SEQ ID NO. 23-24; D3S1358 and SEQ ID NO. 25-26; vWA, SEQ ID NO. 27-28; D5S818 and SEQ ID NO. 29-30; D13S317 and SEQ ID NO. 31-32; D21S11 and SEQ ID NO. 33-34; D2S1338 and SEQ ID NO. 35-36; D12S391 and SEQ ID NO. 37-38; the sequences of the 27 pairs of amplification primers for the Y chromosome STR loci are as follows: DYS392 and SEQ ID NO. 39-40; DYS389I and SEQ ID NO. 41-42; DYS456, SEQ ID NO. 43-44; DYS389II and SEQ ID NO. 45-46; DYS19 and SEQ ID NO. 47-48; DYS460 and SEQ ID NO. 49-50; DYS518 and SEQ ID NO. 51-52; DYS437 and SEQ ID NO. 53-54; DYS576 and SEQ ID NO. 55-56; DYS439 and SEQ ID NO. 57-58; y _ GATA _ H4 and SEQ ID NO. 59-60; DYS533, SEQ ID NO. 61-62; DYS391 and SEQ ID NO. 63-64; DYS627 and SEQ ID NO. 65-66; DYS570, SEQ ID NO. 67-68; DYS635 and SEQ ID NO. 69-70; DYS481 and SEQ ID NO. 71-72; DYS393 and SEQ ID NO. 73-74; DYS390 and SEQ ID NO. 75-76; DYS448 and SEQ ID NO. 77-78; DYS438 and SEQ ID NO. 79-80; DYS458 and SEQ ID NO. 81-82; DYF387S1a and SEQ ID NO. 83-84; DYF387S1b and SEQ ID NO. 85-86; DYS385a and SEQ ID NO. 87-88; DYS385b and SEQ ID NO. 89-90; DYS449, SEQ ID NO. 91-92; 1 amplification primer sequences for the sex recognition loci as follows: amelogenin and SEQ ID NO. 93-94;
dividing the amplification primers of 47 loci into the following five groups, respectively labeling the amplification primers of the five groups by using fluorescent dyes of five different colors, labeling the amplification primers of each group by using the fluorescent dye of the same color, and labeling one amplification primer of each locus by using the fluorescent dye: a first group, DYS392, DYS389I, DYS456, DYS389II, DYS19, DYS460, DYS518, D7S820, TH01, D6S1043; a second group, DYS437, DYS576, DYS439, Y _ GATA _ H4, DYS533, DYS391, DYS627, TPOX, penta E; third group, amelogenin, DYS570, DYS635, DYS481, DYS393, DYS390, DYS448, D16S539, CSF1PO, D19S433; a fourth group, DYS438, DYS458, DYF387S1, DYS385, DYS449, FGA, penta D; a fifth group, D8S1179, D18S51, D3S1358, vWA, D5S818, D13S317, D21S11, D2S1338, D12S391;
the five fluorescent dyes with different colors are marked as 6-FAM, HEX, TAMRA, ROX and NH618;
the working concentration of the primers in the composite amplification kit during amplification is as follows: the working concentration of the 19 pairs of amplification primers of the autosomal STR locus is as follows: D7S820 0.07 mu M, TH01 0.1 mu M, D6S 1043.12 mu M, TPOX 0.09 mu M, penta E0.13 mu M, D16S539 0.11 mu M, CSF1PO 0.13 mu M, D19S4330.18 mu M, FGA 0.09 mu M, penta D0.15 mu M, D8S 1170.2 mu M, D18S510.03 mu M, D3S 1358.04 mu M, vWA 0.05 mu M, D5S818 mu M, D13S317 0.06 mu M, D21S 11.09 mu M, D2S 1330.13 mu M, D12S391 0.16 mu M; the working concentration of the amplification primers for the 27 pairs of Y chromosome STR loci is as follows: DYS392 0.04 mu M, DYS389I 0.03 mu M, DYS456 0.05 mu M, DYS389II0.06 mu M, DYS19 0.05 mu M, DYS460 0.06 mu M, DYS518 0.07 mu M, DYS4370.03 mu M, DYS576 0.04 mu M, DYS439 0.04 mu M, Y _ GATA _ H4.05 mu M, DYS5330.06 mu M, DYS391 0.05 mu M, DYS627 0.07 mu M, DYS627 mu M DYS570 0.05 μ M, DYS635 0.04 μ M, DYS4810.05 μ M, DYS393 0.07 μ M, DYS390 0.06 μ M, DYS448 0.08 μ M, DYS4380.04 μ M, DYS458 0.05 μ M, DYF387S1a 0.06 μ M, DYF387S1b 0.06 μ M, DYS385a0.05 μ M, DYS385b 0.05 μ M and DYS449 0.06 μ M; the working concentration of the 1 pair of amplification primers for the sex recognition locus: 0.03 mu M of Amelogenin;
the amplification reaction program of the composite amplification kit is as follows: 2min at 95 ℃; 30 cycles of 94 ℃ for 5s, 60 ℃ for 45s and 72 ℃ for 45 s; final extension at 60 deg.C for 20min;
the 25 microliter amplification reaction system of the composite amplification kit is as follows: 2.5 multiplied by 10 mul of reaction premixed liquid, 5 mul of 5 multiplied by primer mixture, 8 mul of deionized water and 2 mul of template DNA, wherein the primer mixture is the amplification primer mixture of 47 loci;
the composite amplification kit also comprises reaction premixed solution containing magnesium ions, dNTP and DNA polymerase, allele ladders of 47 loci, DNA standard substances and fluorescent molecular weight internal standard substances.
2. A composite amplification method of 47 loci of human autosomal chromosomes and Y chromosomes is characterized by comprising the following steps:
(1) Sample treatment: extracting the genome DNA of a sample to be detected as an amplification template or directly adopting an extraction-free sample as the amplification template;
(2) Amplifying the sample genomic DNA obtained in the step (1) by using the multiplex amplification kit of claim 1;
(3) Detecting the fluorescent signal of the amplification product;
(4) Fluorescence signal data were collected and analyzed to obtain DNA typing results for 47 loci.
3. The multiplex amplification method of claim 2, wherein the sample to be tested comprises one or more of blood, blood stain, semen stain, bone, hair, saliva stain, sweat, and amniotic fluid containing fetal cells; the extraction-free sample comprises human blood or oral cells collected by one or more carriers of filter paper, blood card, cotton swab and gauze.
4. Use of the multiplex amplification kit of claim 1 for individual identification or paternity testing.
5. Use of the multiplex amplification kit of claim 1 for the construction of a database of human chromosomal DNA loci or for the construction of DNA pedigrees.
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| CN112176067B (en) * | 2019-07-02 | 2022-12-02 | 公安部物证鉴定中心 | Composite amplification system based on Y-STR locus and Y-Indels locus and primer combination used by same |
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| CN115346604B (en) * | 2022-10-20 | 2023-02-10 | 百特元生物科技（北京）有限公司 | DNA sample equilibrium analysis method and device |
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