CN109880912B - Composite amplification kit for 44 human Y chromosome loci and application thereof - Google Patents
Composite amplification kit for 44 human Y chromosome loci and application thereof Download PDFInfo
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Abstract
The invention relates to a 44-person Y chromosome locus composite amplification kit and application thereof. The invention provides a composite amplification system of 44 loci of human Y chromosome, which comprises specific primers for amplifying 44 loci, wherein the 44 loci comprise 41 STR loci of Y chromosome and 3 indels of Y chromosome. The specific primers 44 are subjected to grouped fluorescent labeling by utilizing a six-color fluorescent labeling technology, and efficient, specific and sensitive amplification of the 44 human Y chromosome gene loci is realized through design and optimization of primer sequences and working concentration. 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 44 human Y chromosome gene loci and application thereof.
Background
Short Tandem Repeat sequence loci (STRs) are genetic markers commonly used at present, widely exist in prokaryotic and eukaryotic genomes, and are a type of nucleotide Repeat sequences with more than dozens to one hundred of Repeat units of 2-6 nucleotides. Different numbers of core sequences are in tandem repeats, and the length exhibits polymorphisms. According to the conservative sequences at two ends, a primer is designed for PCR, and then the polymorphism of the STR genetic marker 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, has the advantages of rapidness, high efficiency, accuracy, sensitivity, large information amount and the like due to the similar amplification conditions of all loci, and particularly has great superiority in establishing a DNA database compared with the prior RFLP technology and the like.
The Y chromosome STR genetic marker refers to a short tandem repeat sequence existing in a non-recombination region of a human Y chromosome. At present, 220Y-STR loci are identified and named in various forms, and compared with an autosomal STR genetic marker, most of the Y chromosome STR genetic markers have a complex repetitive structure and contain two or more different repetitive units, and the core repetitive sequences or the repetitive times of individuals in a population are different, so that population genetic polymorphism is formed. The Y chromosome STR genetic marker has three characteristics of male specificity, paternal inheritance, haplotype inheritance and the like, combines the uniqueness with the superiority of STR locus typing detection, and can be used for forensic individual identification, paternity test, DNA family tree construction and the like.
With the development of domestic STR human identification kits and DNA database construction projects and the expansion of the Y-STR database construction market, a kit with higher individual identification rate, more detection loci and better compatibility is urgently needed. The Y STR kit utilizing the five-color fluorescence detection technology cannot meet the requirements of the current market, and the establishment, application and improvement of the six-color fluorescence detection technology can better meet the requirements of the STR locus fluorescence detection kit. Therefore, the field of Y-STR database construction needs to develop a locus amplification system which can realize simultaneous amplification of more loci in one reaction, provide more information, and have better compatibility and faster amplification speed.
Disclosure of Invention
In order to solve the technical problems of limited number of STR loci detected by multiplex amplification simultaneously, poor compatibility of a kit and the like in the prior art, the invention provides a multiplex amplification kit capable of detecting 44 chromosome loci simultaneously and application thereof.
In one aspect, the present invention provides a locus molecular marker for typing human Y chromosomal DNA, wherein the molecular marker comprises the following 44 loci: 41Y chromosome STR loci: DYS456, DYS549, DYS439, DYS19, DYS392, DYS643, DYS447, DYS557, DYS391, DYS388, DYS570, DYS635, DYS448, DYS437, DYS527a, DYS527b, DYS444, DYS393, DYS389I, DYS390, DYS389II, DYS438, DYS576, DYS645, DYS 404S1a, DYS 404S1b, DYS460, DYS458, DYS481, DYS385a, DYS385b, DYS449, DYS596, Y _ GATA _ H4, DYS533, DYS627, DYS518, DYS 387S1a, DYS 387S1b, DYS593, DYS 522; 3Y chromosome Indel: rs199815934, rs771783753 and rs 759551978.
In another aspect, the invention provides a composite amplification kit for 44 loci of human Y chromosome, which comprises the amplification primers for 44 loci.
It is understood by those skilled in the art that the product for multiplex amplification of the 44 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 44 loci and are capable of performing multiplex amplification of the 44 loci are within the scope of the present invention.
Preferably, the sequence of the amplification primer is shown in SEQ ID NO. 1-88: wherein the sequence of the amplification primer is shown as SEQ ID NO. 1-88: wherein, DYS456 and SEQ ID NO. 1-2; DYS549 and SEQ ID NO. 3-4; DYS439 and SEQ ID NO. 5-6; DYS19 and SEQ ID NO. 7-8; DYS392, SEQ ID NO. 9-10; DYS643, SEQ ID NO. 11-12; DYS447, SEQ ID NO. 13-14; DYS557, SEQ ID NO. 15-16; DYS391 and SEQ ID NO. 17-18; DYS388, SEQ ID NO. 19-20; DYS570, SEQ ID NO. 21-22; DYS635 and SEQ ID NO. 23-24; DYS448 and SEQ ID NO. 25-26; DYS437 and SEQ ID NO. 27-28; DYS527a and SEQ ID NO. 29-30; DYS527b and SEQ ID NO. 31-32; DYS444 and SEQ ID NO. 33-34; DYS393 and SEQ ID NO. 35-36; DYS389I, SEQ ID NO. 37-38; DYS390 and SEQ ID NO. 39-40; DYS389II, SEQ ID NO. 41-42; DYS438 and SEQ ID NO. 43-44; DYS576 and SEQ ID NO. 45-46; DYS645 and SEQ ID NO. 47-48; DYF404S1a and SEQ ID NO. 49-50; DYF404S1b and SEQ ID NO. 51-52; DYS460 and SEQ ID NO. 53-54; DYS458 and SEQ ID NO. 55-56; DYS481 and SEQ ID NO. 57-58; DYS385a and SEQ ID NO. 59-60; DYS385b and SEQ ID NO. 61-62; DYS449, SEQ ID NO. 63-64; DYS596 and SEQ ID NO. 65-66; y _ GATA _ H4 and SEQ ID NO. 67-68; DYS533 and SEQ ID NO. 69-70; DYS627 and SEQ ID NO. 71-72; DYS518 and SEQ ID NO. 73-74; DYF387S1a and SEQ ID NO. 75-76; DYF387S1b and SEQ ID NO. 77-78; DYS593, SEQ ID NO. 79-80; DYS522 and SEQ ID NO. 81-82; rs199815934 and SEQ ID NO. 83-84; rs771783753, SEQ ID NO. 85-86; rs759551978 and SEQ ID NO. 87-88.
More preferably, the working concentration of the primers in the multiplex amplification kit during amplification is as follows: working concentration of 41 pairs of Y chromosome STR loci: DYS μ μ 190.1 μ μ μ μ μ M, μ μ μ μ μ μ μ 527 0.12 μ 527 0.12 μ μ 389 0.07 μ 389 0.19 μ μ μ 404S 1.1 μ 404S1 0.1 μ μ 385 0.1 μ 385 0.1 μ GATA _ H40.11 μ μ 387S 1.06 μ 06 μ M; working concentrations of 3 pairs of Y-Indel loci: rs 1998159340.06 μ M, rs 7717837530.08 μ M, rs 7595519780.13 μ M
To achieve simultaneous rapid amplification of the 44 loci, a six-color fluorescent labeling system can be selected to perform fluorescent dye labeling on the amplification primers.
The specific fluorescent dye labeling method is as follows: dividing the amplification primers of the 44 loci into the following five groups, respectively labeling the amplification primers of the five groups by using fluorescent dyes of five different colors, wherein the amplification primers of each group are labeled by the fluorescent dye of the same color, and each locus is provided with one amplification primer labeled by the fluorescent dye: a first group, rs199815934, DYS456, DYS549, DYS439, DYS19, DYS392, DYS643, DYS447, DYS 557; a second group, rs771783753, DYS391, DYS388, DYS570, DYS635, DYS448, DYS437, DYS527a, DYS527b, DYS 444; a third group, rs759551978, DYS393, DYS389I, DYS390, DYS389II, DYS438, DYS576, DYS645, DYF404S1a, DYF404S1 b; a fourth group, DYS460, DYS458, DYS481, DYS385a, DYS385b, DYS449, DYS 596; the fifth group, Y _ GATA _ H4, DYS533, DYS627, DYS518, DYF387S1a, DYF387S1b, DYS593, DYS 522.
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 5FAM or 6FAM fluorescein molecule, the green label is a HEX, VIC or JOE fluorescein molecule, the yellow label is a TAMRA or NED fluorescein molecule, the Red label is a ROX or Texas Red-X fluorescein molecule, and the purple label 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 spectrum similar to that of the fluorescein molecule described above, as desired.
More preferably, the fluorescent dye label is labeled at the 5' end of the primer.
The composite amplification kit can also comprise an amplification primer of an internal quality control fragment, preferably, the internal quality control fragment is IPC60 and IPC 500; more preferably, the amplification primer sequences of the internal quality control fragments IPC60 and IPC500 are respectively shown in SEQ ID NO. 89-90 and SEQ ID NO. 91-92.
As a preferred embodiment, the primer component of the composite amplification kit of the present invention is a mixture of the following primers: amplification primers of 44 loci shown as SEQ ID NO. 1-88 and two internal quality control fragment (IPC) amplification primers shown as SEQ ID NO. 89-92.
SEQ ID NO.89GTCGCCCTTATTCCCTTTTTTGC；
SEQ ID NO.90CGTTTCTGGGTGAGCAAAAACAG；
SEQ ID NO.91GCACTTTTAAAGTTCTGCTATGTGGC；
SEQ ID NO.92TCCAGATTTATCAGCAATAAACCAGCC。
Preferably, the amplification primers of 44 loci and 2 IPCs are divided into the following five groups: the first group, IPC60, rs199815934, DYS456, DYS549, DYS439, DYS19, DYS392, DYS643, DYS447, DYS557, IPC 500; a second group, rs771783753, DYS391, DYS388, DYS570, DYS635, DYS448, DYS437, DYS527a, DYS527b, DYS 444; a third group, rs759551978, DYS393, DYS389I, DYS390, DYS389II, DYS438, DYS576, DYS645, DYF404S1a, DYF404S1 b; a fourth group, DYS460, DYS458, DYS481, DYS385a, DYS385b, DYS449, DYS 596; the fifth group, Y _ GATA _ H4, DYS533, DYS627, DYS518, DYF387S1a, DYF387S1b, DYS593, DYS 522.
The amplification reaction program of the composite amplification kit is as follows: root at 95-98 deg.c for 1-5 min; 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 20 min.
When the 44 loci composite amplification kit is used for amplification, 25 mul of amplification reaction system is as follows: 2.5 times of reaction premix 10 μ l, 5 times of primer mixture 5 μ l, 8 μ l of deionized water, and 2 μ l of template DNA, wherein the primer mixture is the amplification primer mixture of 44 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 44 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 (25ul) typically requires 2U to 4U of Taq DNA polymerase.
The reaction premix comprises dNTP, DNA polymerase and buffer solution of the DNA polymerase; wherein, the buffer solution of the DNA polymerase can comprise: 50mM KCl, 10mM Tris HCl (pH8.3, 25 ℃), 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 which are prepared by mixing dNTP, DNA polymerase and buffer of DNA polymerase into 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 44 loci of human 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 as SEQ ID NO. 1-88;
(3) detecting the fluorescent signal of the amplification product;
(4) fluorescence signal data was collected and analyzed to obtain DNA typing results for 44 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 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 4 ng.
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 ABI 3130, 3100, 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 length and is used for calculating the length of PCR amplification product segments, thereby being capable of judging the genotyping and the allele ladder comparison.
The collected data such as fluorescence signals can be analyzed on data analysis software such as GeneMapper, GeneMarker, GeneScan and the like, and finally the STR genotyping map and data are obtained.
In addition, the invention also provides an amplification primer group, which comprises 44 pairs of primers with sequences shown as SEQ ID NO. 1-88.
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 for 44 human chromosome locus combinations
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 have analyzed genetic polymorphisms of chromosomes of a plurality of loci in common for different individuals, and have finally determined combinations of 44 chromosomal loci in a combination of 62 loci including DYS446, DYS510, DYS622, DYS443, DYS587, GATA _ a10, DYS520, DYS552, DYS531, DYS459ab, DYS508, DYS617, DYS713, DYS630, DYS626 and DYS526ab, on the basis of sufficient analysis of loci used in conventional kits: 41Y chromosome STR loci: DYS456, DYS549, DYS439, DYS19, DYS392, DYS643, DYS447, DYS557, DYS391, DYS388, DYS570, DYS635, DYS448, DYS437, DYS527a, DYS527b, DYS444, DYS393, DYS389I, DYS390, DYS389II, DYS438, DYS576, DYS645, DYS 404S1a, DYS 404S1b, DYS460, DYS458, DYS481, DYS385a, DYS385b, DYS449, DYS596, Y _ GATA _ H4, DYS533, DYS627, DYS518, DYS 387S1a, DYS 387S1b, DYS593, DYS 522; 3Y chromosome Indel: rs199815934, rs771783753 and rs 759551978. Wherein, on the basis of the golden eye 30Y kit, the kit increases 14 highly genetic polymorphic loci. Therefore, the kit disclosed by the invention breaks through the maximum composite amplification which can be realized at present and detects the number of loci simultaneously, and has the advantages of obviously improved individual recognition capability and non-paternal exclusion rate. The arrangement positions of the 44 loci on the electropherogram 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 traditional six-color fluorescence technologies commonly used at present 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 of low fluorescence efficiency due to the long wavelength of excitation light of a long-distance genetic analyzer of maximum absorption light of fluorescein molecules. 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, comprehensively considering factors such as optimal amplification fragment length and the like, dividing 44 loci into five groups, wherein each group is respectively marked by different fluorescein, amplification products of each locus in each group are separated according to length difference, and two loci cannot be overlapped. By reasonably matching the PCR amplification efficiency of the gene loci and the fluorescence efficiency of the fluorescein marker, the amplified fragments have approximate RFU values in capillary electrophoresis analysis, and the balance is obviously improved.
The following preferred groups are obtained by a large number of optimization experiments: the first group, IPC60, rs199815934, DYS456, DYS549, DYS439, DYS19, DYS392, DYS643, DYS447, DYS557, IPC 500; a second group, rs771783753, DYS391, DYS388, DYS570, DYS635, DYS448, DYS437, DYS527a, DYS527b, DYS 444; a third group, rs759551978, DYS393, DYS389I, DYS390, DYS389II, DYS438, DYS576, DYS645, DYF404S1a, DYF404S1 b; a fourth group, DYS460, DYS458, DYS481, DYS385a, DYS385b, DYS449, DYS 596; the fifth group, Y _ GATA _ H4, DYS533, DYS627, DYS518, DYF387S1a, DYF387S1b, DYS593, DYS 522.
Specific primers were then designed for the 44 loci described above flanking their repeat sequences and Indel insertion/deletion sites, respectively. The basic principle of primer design is as follows: the annealing temperature of each primer is close to 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 were performed using primer pairs for each group 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 44 loci, continuously optimizing 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 44 pairs of primers in a primer mixture through a large amount of 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 41Y chromosome STR loci and 3Y chromosome Indel loci for the first time, realizes the simultaneous high-efficiency, specific and sensitive amplification of 44 loci by ingenious primer sequences and use concentration design and improved six-color fluorescence labeling technology, and is a composite amplification system which can realize the maximum number of loci simultaneously amplified by the currently reported human Y chromosome STR typing technology;
(2) the combination of 44 chromosome loci provided by the invention adds a plurality of loci with high genetic polymorphism on the basis of all loci used by the existing Y-STR typing detection reagent at home and abroad, thereby obviously improving the individual identification capability of detection;
(3) the combination of 44 chromosome loci provided by the invention integrates all loci adopted by the current Y-STR typing detection reagent at home and abroad, has good analysis data compatibility, can be compatible with all existing 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 44 chromosome loci provided by the invention is higher than the information amount obtained by simultaneously using 2 to 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, male samples have no non-specific amplification, female samples have no amplification, and the temperature tolerance range is wider, so that different PCR amplification instruments can obtain better amplification results;
(7) the multiplex amplification system provided by the invention can be widely applied to the construction of a Y-STR haplotype database, and is also suitable for field case detection, in particular to 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 44 loci provided by 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 44 loci of the 9948DNA composite amplification kit provided by the present invention.
FIG. 4 is a typing map of 44 loci of 9948DNA in comparative example 1.
FIG. 5 is a typing map of 44 loci of 9948DNA in comparative example 2.
FIG. 6 is a genotyping map of 44 loci of suspected progenitors identified by genetic relationship in example 4.
FIG. 7 is a typing map of the suspected grandchild 44 loci identified by the genetic relationship in example 4.
FIG. 8 is a typing map of 44 loci of sample 1 in the database construction in example 5.
FIG. 9 is a typing map of 44 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 144 development of multiplex amplification kit for chromosomal loci
1. Multiplex amplification primer design for 44 chromosome loci
For a multiplex amplification system, especially for the multiplex amplification system of 44 loci in the invention, the requirements for specificity of primer sequence, secondary structure, stability of combination with target sequence, amplification efficiency and the like are extremely high, and through continuous cyclic experiments of amplification-optimization-amplification, 44 pairs of primers for specifically and efficiently amplifying 44 loci are obtained, and the specific sequences are shown in table 1:
amplification primers for Table 144 loci and 2 IPCs
Carrying out fluorescent labeling on the primers with different colors according to the following grouping modes of corresponding loci: the first group, IPC60, rs199815934, DYS456, DYS549, DYS439, DYS19, DYS392, DYS643, DYS447, DYS557, IPC 500; a second group, rs771783753, DYS391, DYS388, DYS570, DYS635, DYS448, DYS437, DYS527a, DYS527b, DYS 444; a third group, rs759551978, DYS393, DYS389I, DYS390, DYS389II, DYS438, DYS576, DYS645, DYF404S1a, DYF404S1 b; a fourth group, DYS460, DYS458, DYS481, DYS385a, DYS385b, DYS449, DYS 596; the fifth group, Y _ GATA _ H4, DYS533, DYS627, DYS518, DYF387S1a, DYF387S1b, DYS593, DYS 522. 1-5 groups were labeled with blue, green, yellow, red and violet fluorescein, respectively. Only one strand of each primer pair is labeled, with the label being at the 5' end of the primer. Blue marker selection 6-FAM, green marker selection HEX, yellow marker selection TAMRA, red marker selection ROX, purple marker selection NH 618.
2. Optimization of primer concentration in multiplex amplification reaction system
For a composite amplification system for amplifying 44 loci simultaneously, in order to realize higher amplification equilibrium degree, the concentration ratio of 44 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: DYS μ μ 190.1 μ μ μ μ μ M, μ μ μ μ μ μ μ 527 0.12 μ 527 0.12 μ μ 389 0.07 μ 389 0.19 μ μ μ 404S 1.1 μ 404S1 0.1 μ μ 385 0.1 μ 385 0.1 μ GATA _ H40.11 μ μ 387S 1.06 μ 06 μ M; rs 1998159340.06 μ M, rs 7717837530.08 μ M, rs 7595519780.13 μ M, IPC 600.03 μ M, IPC 5000.1 μ M.
The 44 chromosome locus multiplex amplification kit consists of the components shown in the table 2.
TABLE 2 composite amplification kit composition
Wherein the primer mixture is a mixture of 44 pairs of primers with sequences shown as SEQ ID NO. 1-88 and 2 pairs of IPC amplification primers shown as SEQ ID NO. 89-92.
Example establishment of a Complex amplification System and procedure for 244 chromosomal loci
The annealing temperature and cycle number were optimized for the amplification primers of 44 loci, 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 44Y chromosome loci on a 9948 cell strain. A template DNA derived from 9948 cell line was extracted by the chelex 100 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 chelex 100 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 well with shaking to suspend the chelex well, and 195. mu.l of 5% chelex 100(l00-200mesh, available from Bio Rad) was added to each tube, followed by 5. mu.l of proteinase K (20mg/ml, available from Tiangen Biochemical technology 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 44 pairs of primers with sequences shown as SEQ ID NO. 1-88; the reaction premix comprises: 50mM KCI, l0mM Tris HCI (pH8.3, 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 hot start DNA polymerase, antibody blocking modification or chemical modification is possible, and 2U to 4U of Taq DNA polymerase is required for each amplification system (25 ul).
(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 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, adding 10 μ l of each tube, adding 1 μ l of amplification product or allele-specific ladder (ladder) (shown in FIG. 2), and centrifuging for a short time to collect the liquid at the bottom of the tube.
(3) The samples were 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 was placed in the sample tray of a gene analyzer, the instrument parameters (sample injection voltage 3kV, sample injection time 4 seconds) were set, and the electrophoresis detection was 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. 3, and the typing information of the 44Y chromosome loci of the 9948 cell strain obtained by using the kit provided by the invention is consistent with the information in the database, which proves that the kit provided by the invention can realize the accurate detection of the 44 chromosome loci at one time.
Comparative example 1 amplification Using primers of different sequences
Typing of 44 chromosomal loci of 9948 cell line was carried out using the composite amplification system and procedure in examples 1 and 2 and the detection method in example 3 by replacing the primers of the DYS456, DYS448 and DYS645 loci, respectively, with the primer sequences in Table 4, and as a result, as shown in FIG. 4, non-specific amplification similar to n-2 occurred between the main peak of DYS456 and the stutter peak; obvious non-specific amplification appears at the front 50bp of a data analysis area, and the major peak of DYS448 is obviously lower; the major peak of DYS645 showed a more pronounced tailing phenomenon (circled in the figure). The results show that the primer pairs in Table 4 used for substitution may cause adverse effects in the DNA typing process, are not suitable for the primer combination of the present invention, and the influence of the primer sequence on the detection result of the kit is large.
TABLE 4 primer sequences used in comparative example 1
Comparative example 2 Effect of variation in primer concentration
The primer concentrations of the loci DYS481 and DYS627 are respectively replaced by the primer concentrations in Table 5, and the detection method in example 3 is used to carry out typing on 44 chromosome loci of 9948 cell strains by using the composite amplification system and the program in examples 1 and 2, and the results are shown in FIG. 5, which shows that the adjustment of the primer concentrations can obviously influence the dye sets and the overall balance of the kit. After the primer concentration of DYS481 is adjusted, the product peak height of the gene locus in an electrophoretogram is obviously changed and is more than 2 times higher than that of the adjacent DYS458, and the balance cannot meet the requirement of a kit; after the primer concentration of DYS627 is adjusted, the peak height is correspondingly and obviously reduced, 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 |
| DYS481 | 0.25μM |
| DYS627 | 0.03μM |
Example 4 genetic relationship identification Using multiplex amplification kit
The kit provided by the invention is used for carrying out composite amplification of 44Y chromosome loci on 2 blood card samples from suspected grandfather. PCR amplification is carried out by adopting a method of direct amplification of 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. 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. Blood samples were each punched out of blood cards from grandfather two using a punch of 1.2mm diameter, taking care that the position of the punch was sufficiently permeable to the blood sample and 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 a buffer solution and a primer mixture, preparing a mixed solution according to the following table, shaking, mixing uniformly, subpackaging into PCR reaction tubes, adding template DNA, and preparing into a10 mu l reaction system (table 6).
TABLE 6 PCR amplification reaction System
Wherein the primer mixture is a mixture of 44 pairs of primers with sequences shown as SEQ ID NO. 1-88; the reaction premix comprises: 50mM KCl, l0mM Tris HCl (pH8.3, 25 ℃), 2.0mM MgCl 2 0.1mg/ml BSA (bovine serum albumin) and 0.2mM each of dNTPs. dNTP is four kinds of deoxyribonucleotides (dATP, dTTP, dCTP, dGTP) and the like molar mixture, and reaction required Taq DNA polymerase, the Taq DNA polymerase used is hot start DNA polymerase, antibody blocking modification or chemical modification can be performed, each amplification system (10 μ l) needs 1U to 2U of hot start 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 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 the allelic ladder (ladder) (shown in FIG. 2) was added to the tubes containing the above mixture as the amplification product, and the mixture was collected in the bottom of the tube by brief centrifugation.
(3) The samples were 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 was placed in the sample tray of a gene analyzer, the instrument parameters (sample injection voltage 3kV, sample injection time 4 seconds) were set, and the electrophoresis detection was 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 ancestor) and fig. 7 (suspected grandchild), the typing information of the 44Y chromosome loci of the suspected grandchild obtained by the kit provided by the present invention is compared, and the typing of the suspected grandchild on the 44Y chromosome loci is consistent, and conforms to the genetic rule. In combination with the actual situation, a genetic relationship determination supporting the suspected grandchild to be grandchild may be made.
Example 5 judicial identification Using multiplex amplification kit
The kit provided by the invention is used for carrying out composite amplification on 44Y chromosome loci on 2 blood card samples commonly used in the market. PCR amplification is carried out by adopting a method of direct amplification of 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. 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. Blood slices are punched on 2 blood card samples respectively by a puncher with the diameter of 1.2mm, and the positions for punching the blood slices are required 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 following table, shaking, uniformly mixing, subpackaging into PCR reaction tubes, adding the template DNA, and preparing into a10 mu l reaction system (table 7).
TABLE 7 PCR amplification reaction System
Wherein the primer mixture is a mixture of 44 pairs of primers with sequences shown as SEQ ID No. 1-88; the reaction premix comprises: 50mM KCl, l0mM Tris HCl (pH8.3, 25 ℃), 2.0mM MgCl 2 0.1mg/ml BSA (bovine serum albumin) and 0.2mM each of dNTPs. dNTP is a mixture of four kinds of deoxyribonucleotides (dATP, dTTP, dCTP, dGTP) in equal mole, and reactionThe required Taq DNA polymerase is hot-start Taq DNA polymerase, the antibody blocking modification or the chemical modification is available, and 1U to 2U of hot-start Taq DNA polymerase is required for each amplification system (10 mu l). (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 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 the allelic ladder (ladder) (shown in FIG. 2) was added to the tubes containing the above mixture as the amplification product, and the mixture was collected in the bottom of the tube by brief centrifugation.
(3) The samples were 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 was placed in the sample tray of a gene analyzer, the instrument parameters (sample injection voltage 3kV, sample injection time 4 seconds) were set, and the electrophoresis detection was 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 of the test material 1 and the test material 2 are respectively shown in fig. 8 and fig. 9, and the typing information of 44Y chromosome loci of the sample constructed by the Y chromosome STR database obtained by using the kit provided by the invention is used for deriving the typing result and inputting the typing result into the database.
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 therefore intended that such modifications and variations be included within the purview of the appended claims.
Sequence listing
<110> Kyoto cognitive technology (Beijing) Ltd
Composite amplification kit for <120> 44 human Y chromosome loci and application thereof
<160> 92
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtctgttgtg ggaccttgtg ataatg 26
<210> 2
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gttctctaga gggacagaac taatgg 26
<210> 3
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atagcaatta ggtaggtaaa gaggaa 26
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tttttttggt ggcataagtg gtaatg 26
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cttcctaggt tttcttctcg agttgt 26
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggaattcttt tacccatcat ctctttac 28
<210> 7
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctaggtatga gatcaaattg actgtgat 28
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caggagtaat acttcgggcc at 22
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
catctccatc catgttgctc ca 22
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
agtctggaaa tatctcaaag aagtcaaaac 30
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgtatttgcc aaagcttaat cttgaaattt 30
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gggataaaag actacatatt gggtaaaatg 30
<210> 13
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gacatgcctg tgctacaact tca 23
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tcagagagac agatgcattt ctg 23
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
<210> 16
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cacagtctct cccgtgatgg aac 23
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
catacaccca tatctgtctg tct 23
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gccatagagg gataggtagg cag 23
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
catgtgagtt agccgtttag cga 23
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
<210> 21
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ggttatgaaa cgtaaaatga atgatgac 28
<210> 22
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
attcagcata gtcaagaaac cagac 25
<210> 23
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gatccataga tatcaagagt gtctcact 28
<210> 24
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
caaaattcac agttggaaaa atgtggaa 28
<210> 25
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tccaaataaa gaacagagaa gtgtcaaa 28
<210> 26
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gttggagacc ttttcttcct taacgt 26
<210> 27
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
atcatctgtg aatgacaggg tcttcc 26
<210> 28
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ggctgcagtg agctgtgatt atg 23
<210> 29
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aaagaaagga aggaaggaag caagg 25
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgagagtaaa aattaacaat tttaacagct 30
<210> 31
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
aaagaaagga aggaaggaag caagg 25
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tgagagtaaa aattaacaat tttaacagct 30
<210> 33
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
catcaacata gaatgaaagg tgtgaa 26
<210> 34
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tagaatgtta aagaaaatca taaggcagag 30
<210> 35
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tttgtcattc ctaatgtggt cttctacttg 30
<210> 36
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
aaaaaatgag gtatgtctca tagaaaagac 30
<210> 37
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tccaactctc atctgtatta tctatgtatc tg 32
<210> 38
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
acttgaggaa cacaattatc cctgagtagc 30
<210> 39
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
cacatttttg ggccctgcat tttgg 25
<210> 40
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
atgagtggga gaaatggatg acagt 25
<210> 41
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ctctcatctg tattatctat gtgtgtgtc 29
<210> 42
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
acttgaggaa cacaattatc cctgagtagc 30
<210> 43
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ccattagagc atcacagtaa taactgat 28
<210> 44
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
caacatggtg aaaccctgtc tcta 24
<210> 45
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
<210> 46
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
atgggaaaaa cccaacacca tg 22
<210> 47
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
ggtacagttc tttggttttg gttac 25
<210> 48
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ccattgaccc aagaagcata ctacc 25
<210> 49
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ggcctaatgc acaagaactc taagt 25
<210> 50
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ccaggtattc tggttgaggc taag 24
<210> 51
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
ggcctaatgc acaagaactc taagt 25
<210> 52
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ccaggtattc tggttgaggc taag 24
<210> 53
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
gcacaagaat accagaggaa tctg 24
<210> 54
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
cctctgccta tcatttatta tgtatttgtc 30
<210> 55
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
cagactgagc aacaggaatg aaact 25
<210> 56
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
gttctggcat tacaagcatg agc 23
<210> 57
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
tgtgtgtgtg atattctgtt tgtgtgtg 28
<210> 58
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
caagaagagc cacacagaca gct 23
<210> 59
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
catgggtgac agagctagac ac 22
<210> 60
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
aatctatcta ttccaattac atagtcctcc 30
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
catgggtgac agagctagac ac 22
<210> 62
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
aatctatcta ttccaattac atagtcctcc 30
<210> 63
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gctagagatt cttggagtct ctcaa 25
<210> 64
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ctggaagtgg agtttgctgt aa 22
<210> 65
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
cacccttatt gtacttctgt gataagct 28
<210> 66
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
gcccaaagtt cttaacttcc ttttcaag 28
<210> 67
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ccaaatttaa gatagataga tagatcta 28
<210> 68
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ctatctattc atccatctaa tctatcca 28
<210> 69
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
cttctaccta tcatctttct agctagc 27
<210> 70
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
gctgtaagta gagatcacca atgaaatg 28
<210> 71
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
<210> 72
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
catccttcct tcttaccttc tttacttc 28
<210> 73
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
<210> 74
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
tcttcagctc ttaccatggg tga 23
<210> 75
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ctgggtgaca gagctagatt ccatt 25
<210> 76
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
caaagttgca actgttttac agcac 25
<210> 77
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
ctgggtgaca gagctagatt ccatt 25
<210> 78
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
caaagttgca actgttttac agcac 25
<210> 79
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
cttgaaccca ggaagcagac cttac 25
<210> 80
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ctaggtggga acttctatag gcaac 25
<210> 81
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gagagagaga tgatggatag ataaatagat 30
<210> 82
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
acaaatttgt cctcactggt ttctt 25
<210> 83
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
gatttaaact ctctgaatca ggcac 25
<210> 84
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
actgatacct ttgtttctgt tcatt 25
<210> 85
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
ataatagggg aggcttgtca ctaat 25
<210> 86
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
caaggtcaca gagattgtca acatt 25
<210> 87
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gacagttatc agtttgaaat tattttttca 30
<210> 88
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
taactcacca aaggaatgca catct 25
<210> 89
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
gtcgccctta ttcccttttt tgc 23
<210> 90
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
cgtttctggg tgagcaaaaa cag 23
<210> 91
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
gcacttttaa agttctgcta tgtggc 26
<210> 92
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
tccagattta tcagcaataa accagcc 27
Claims (17)
1. A composite amplification kit for 44 loci of human Y chromosome is characterized by comprising amplification primers of 44 loci;
the sequence of the amplification primer is shown as SEQ ID NO. 1-88: wherein, DYS456 and SEQ ID NO. 1-2; DYS549 and SEQ ID NO. 3-4; DYS439 and SEQ ID NO. 5-6; DYS19, SEQ ID NO. 7-8; DYS392, SEQ ID NO. 9-10; DYS643, SEQ ID NO. 11-12; DYS447, SEQ ID NO. 13-14; DYS557, SEQ ID NO. 15-16; DYS391 and SEQ ID NO. 17-18; DYS388, SEQ ID NO. 19-20; DYS570, SEQ ID NO. 21-22; DYS635 and SEQ ID NO. 23-24; DYS448 and SEQ ID NO. 25-26; DYS437 and SEQ ID NO. 27-28; DYS527a and SEQ ID NO. 29-30; DYS527b and SEQ ID NO. 31-32; DYS444 and SEQ ID NO. 33-34; DYS393 and SEQ ID NO. 35-36; DYS389I, SEQ ID NO. 37-38; DYS390 and SEQ ID NO. 39-40; DYS389II, SEQ ID NO. 41-42; DYS438 and SEQ ID NO. 43-44; DYS576 and SEQ ID NO. 45-46; DYS645 and SEQ ID NO. 47-48; DYF404S1a and SEQ ID NO. 49-50; DYF404S1b and SEQ ID NO. 51-52; DYS460 and SEQ ID NO. 53-54; DYS458 and SEQ ID NO. 55-56; DYS481 and SEQ ID NO. 57-58; DYS385a and SEQ ID NO. 59-60; DYS385b and SEQ ID NO. 61-62; DYS449, SEQ ID NO. 63-64; DYS596 and SEQ ID NO. 65-66; y _ GATA _ H4 and SEQ ID NO. 67-68; DYS533 and SEQ ID NO. 69-70; DYS627 and SEQ ID NO. 71-72; DYS518 and SEQ ID NO. 73-74; DYF387S1a and SEQ ID NO. 75-76; DYF387S1b and SEQ ID NO. 77-78; DYS593 and SEQ ID NO. 79-80; DYS522 and SEQ ID NO. 81-82; rs199815934 and SEQ ID NO. 83-84; rs771783753, SEQ ID NO. 85-86; rs759551978 and SEQ ID NO. 87-88.
2. The multiplex amplification kit of claim 1, wherein the primers for the 44 loci of the multiplex amplification kit are amplified at working concentrations as follows: DYS [ mu ] n [ 0.12 ] n [ mu ] n [ 5 [ mu ] n [ mu ] M [ 1 [ mu ] M [ mu ] p [ mu; rs 1998159340.06 mu M, rs 7717837530.08 mu M, rs 7595519780.13 mu M.
3. The multiplex amplification kit according to any one of claims 1 to 2, wherein the amplification primers carry a fluorescent dye label.
4. The multiplex amplification kit of claim 3, wherein the amplification primers for 44 loci are divided into five groups, and the five groups of amplification primers are labeled with five different colors of fluorescent dyes, respectively, wherein the amplification primers in each group are labeled with the same color of fluorescent dye, and one amplification primer for each locus is labeled with a fluorescent dye: a first group, rs199815934, DYS456, DYS549, DYS439, DYS19, DYS392, DYS643, DYS447, DYS 557; a second group, rs771783753, DYS391, DYS388, DYS570, DYS635, DYS448, DYS437, DYS527a, DYS527b, DYS 444; a third group, rs759551978, DYS393, DYS389I, DYS390, DYS389II, DYS438, DYS576, DYS645, DYF404S1a, DYF404S1 b; a fourth group, DYS460, DYS458, DYS481, DYS385a, DYS385b, DYS449, DYS 596; the fifth group, Y _ GATA _ H4, DYS533, DYS627, DYS518, DYF387S1a, DYF387S1b, DYS593, DYS 522.
5. The multiplex amplification kit of claim 4, wherein said five fluorescent dye labels of different colors are blue, green, yellow, red and purple fluorescent labels, respectively.
6. The multiplex amplification kit of claim 5, 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 label is a TAMRA or NED fluorescein molecule, the Red label is a ROX or Texas Red-X fluorescein molecule, and the purple label is a NH618 fluorescein molecule.
7. The multiplex amplification kit of claim 6, wherein said five different color fluorescent dyes are labeled 6-FAM, HEX, TAMRA, ROX, NH 618.
8. The composite amplification kit as claimed in any one of claims 1 to 2, further comprising amplification primers of internal quality control fragments IPC60 and IPC 500; the amplification primer sequences of the internal quality control fragments IPC60 and IPC500 are respectively shown in SEQ ID NO. 89-90 and SEQ ID NO. 91-92.
9. The multiplex amplification kit according to any one of claims 1 to 2, wherein the amplification reaction is performed by the following procedure: 1 min-5 min at the temperature of 95-98 ℃; 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 ℃ for 5-30 min.
10. The multiplex amplification kit according to claim 9, wherein 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 20 min.
11. The composite amplification kit according to any one of claims 1 to 2, wherein a 25 μ l amplification reaction system is as follows: 10 mul of 2.5 multiplied reaction premixed liquid, 5 mul of 5 multiplied primer mixture, 8 mul of deionized water and 2 mul of template DNA, wherein the primer mixture is the amplification primer mixture of 44 loci.
12. The multiplex amplification kit of any one of claims 1 to 2, further comprising a reaction premix containing magnesium ions, dNTPs, DNA polymerase, an allele ladder at 44 loci, a DNA standard, and a fluorescent molecular weight internal standard.
13. A method for multiplex amplification of 44 loci of human Y chromosome using the multiplex amplification kit of any one of claims 1 to 12, comprising the steps of:
(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 as SEQ ID NO. 1-88;
(3) detecting the fluorescent signal of the amplification product;
(4) fluorescence signal data was collected and analyzed to obtain DNA typing results for 44 loci.
14. The multiplex amplification method of claim 13, wherein said sample comprises one or more of blood, blood spots, semen spots, bone, hair, saliva spots, sweat, and amniotic fluid containing fetal cells; the non-extracted sample comprises human blood or oral cells collected by one or more carriers of filter paper, blood card, cotton swab and gauze.
15. A primer mixture for amplifying 44 loci of human Y chromosome is characterized by comprising 44 pairs of primers with sequences shown as SEQ ID NO. 1-88.
16. Use of the multiplex amplification kit of any one of claims 1 to 10 or the primer mixture of claim 15 for individual identification or paternity testing.
17. Use of the multiplex amplification kit of any one of claims 1 to 10 or the primer mixture of claim 15 for the construction of a database of human chromosomal DNA loci or for the construction of DNA pedigrees.
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