CN112852970A - Primer group and kit for simultaneously amplifying 37 human Y-STR loci and application of primer group and kit - Google Patents
Primer group and kit for simultaneously amplifying 37 human Y-STR loci and application of primer group and kit Download PDFInfo
- Publication number
- CN112852970A CN112852970A CN202011621943.8A CN202011621943A CN112852970A CN 112852970 A CN112852970 A CN 112852970A CN 202011621943 A CN202011621943 A CN 202011621943A CN 112852970 A CN112852970 A CN 112852970A
- Authority
- CN
- China
- Prior art keywords
- seq
- nucleotide sequence
- primer
- amplifying
- independently
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000003321 amplification Effects 0.000 claims abstract description 62
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 58
- 230000002068 genetic effect Effects 0.000 claims abstract description 14
- 210000000349 chromosome Anatomy 0.000 claims abstract description 9
- 238000010276 construction Methods 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims description 205
- 125000003729 nucleotide group Chemical group 0.000 claims description 205
- 238000011144 upstream manufacturing Methods 0.000 claims description 103
- 238000012408 PCR amplification Methods 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 17
- 229910021641 deionized water Inorganic materials 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 239000007850 fluorescent dye Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 210000002593 Y chromosome Anatomy 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 5
- 239000007983 Tris buffer Substances 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 3
- 108010006785 Taq Polymerase Proteins 0.000 claims description 3
- 229960003237 betaine Drugs 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 15
- 239000000463 material Substances 0.000 abstract description 8
- 108020004414 DNA Proteins 0.000 description 103
- 108091092878 Microsatellite Proteins 0.000 description 26
- 239000008280 blood Substances 0.000 description 11
- 210000004369 blood Anatomy 0.000 description 11
- 108700028369 Alleles Proteins 0.000 description 9
- 238000001962 electrophoresis Methods 0.000 description 9
- 238000003205 genotyping method Methods 0.000 description 9
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
- 239000013641 positive control Substances 0.000 description 7
- 210000004209 hair Anatomy 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 101150084399 37 gene Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001215 fluorescent labelling Methods 0.000 description 3
- 210000003780 hair follicle Anatomy 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 210000003296 saliva Anatomy 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000282 nail Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000000582 semen Anatomy 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001909 effect on DNA Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 230000008775 paternal effect Effects 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a primer group for simultaneously amplifying 37 human Y-STR loci, a kit and application thereof, belonging to the technical field of molecular genetics. In the invention, the 37Y-STR loci comprise 36Y chromosome STR loci and 1Y-indel locus; the invention can realize the simultaneous amplification of 37 loci in one reaction, fully meets the compatibility of the current public security DNA database comparison, reduces the amplification time, improves the identification efficiency and the material detection adaptability of the kit, and can meet the requirements of the current multifunctional STR identification of forensic identity identification, forensic DNA database construction and judicial genetic relationship identification.
Description
Technical Field
The invention relates to the technical field of molecular genetics, in particular to a primer group and a kit for simultaneously amplifying 37 human Y-STR loci and application thereof.
Background
STR (short tandem repeats), also known as microsatellite sequences, is a short tandem repeat that is present in large amounts in human genomic DNA and has a repeat unit of 2-6 nucleotides. Because of the high polymorphism and stability, and the much smaller length of amplified product (less than 500bp) compared to the AMP-FLP and VNTR isotyping methods, STR genotyping requires less template quality and allows analysis even with degraded DNA templates. In addition, STR typing is suitable for DNA purified by a variety of DNA purification methods, which often yield DNA in amounts insufficient for Southern blot analysis. In view of the above characteristics, STR typing techniques have been widely applied to forensic identification.
The forensic DNA database integrates the elements of modern DNA inspection technology, information technology, network technology, scientific management and the like. The core of the forensic DNA database lies in the recorded STR data information, and a perfect forensic DNA database should contain an autosomal STR database and a Y chromosome STR database. The Y chromosome STR data provides investigation clues for cases, reduces investigation range, can predict regions, ethnicities or even surnames where criminals may exist, and the autosome STR data is used for screening and confirming final criminal individuals.
The currently published 20Y-STR core loci (20 loci, including DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y GATAH4, DYS481, DYS533, DYS576) and 15Y-STR preferred loci (15 loci, including DYS, DYS460, DYS549, DYS387S1a/b, DYS449, DYS518, DYS627, DYS570, DYS527a/b, DYS447, DYS444, DYS557, DYS) have a higher requirement for the number of loci, specificity and sensitivity of the kit by DNA forensic laboratory.
Disclosure of Invention
The invention aims to provide a primer group and a kit for amplifying 37 human Y-STR loci simultaneously and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer group for simultaneously amplifying 37Y-STR loci of a human, wherein the 37Y-STR loci comprise 36Y chromosome STR loci and 1Y-indel locus; the 36Y chromosome STR loci are respectively: DYS438, DYS389I, DYS448, DYS389II, DYS19, Y _ GATA _ H4, DYS518, DYS460, DYS458, DYS437, DYS439, DYS643, dysf 387S1a/b, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS449, DYS596, DYS456, DYS576, DYS549, DYS570, DYS481, DYS392, DYS627, DYS388, DYS447, DYS557, DYS527a/b, DYS533 and DYS 444;
the nucleotide sequence of the upstream primer for amplifying DYS438 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS389I is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS389II is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS448 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS19 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying Y _ GATA _ H4 is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS518 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS460 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS458 is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS437 is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS439 is shown as SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22; the nucleotide sequence of the upstream primer for amplifying DYS643 is shown as SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24; the nucleotide sequence of the upstream primer for amplifying DYF387S1a/b is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 26 is shown; the nucleotide sequence of the upstream primer for amplifying DYS385a/b is shown as SEQ ID NO: 27, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 28 is shown; the nucleotide sequence of the upstream primer for amplifying DYS393 is shown in SEQ ID NO: 29, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 30 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS391 is shown as SEQ ID NO: 31, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 32 is shown; the nucleotide sequence of the upstream primer for amplifying DYS390 is shown as SEQ ID NO: 33, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 34; the nucleotide sequence of the upstream primer for amplifying DYS635 is shown as SEQ ID NO: 35, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 36 is shown; the nucleotide sequence of the upstream primer for amplifying DYS449 is shown as SEQ ID NO: 37, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 38; the nucleotide sequence of the upstream primer for amplifying DYS596 is shown as SEQ ID NO: 39, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 40 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS456 is shown as SEQ ID NO: 41, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 42 is shown; the nucleotide sequence of the upstream primer for amplifying DYS576 is shown as SEQ ID NO: 43, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 44 is shown; the nucleotide sequence of the upstream primer for amplifying DYS549 is shown as SEQ ID NO: 45, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 46; the nucleotide sequence of the upstream primer for amplifying DYS570 is shown as SEQ ID NO: 47, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 48 is shown; the nucleotide sequence of the upstream primer for amplifying DYS481 is shown as SEQ ID NO: 49, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 50 is shown; the nucleotide sequence of the upstream primer for amplifying DYS392 is shown as SEQ ID NO: 51, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 52; the nucleotide sequence of the upstream primer for amplifying DYS627 is shown as SEQ ID NO: 53, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 54 is shown; the nucleotide sequence of the upstream primer for amplifying the Y-indel is shown as SEQ ID NO: 55, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 56 is shown; the nucleotide sequence of the upstream primer for amplifying DYS388 is shown as SEQ ID NO: 57, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 58; the nucleotide sequence of the upstream primer for amplifying DYS447 is shown as SEQ ID NO: 59, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 60 is shown; the nucleotide sequence of the upstream primer for amplifying DYS557 is shown as SEQ ID NO: 61, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 62; the nucleotide sequence of the upstream primer for amplifying DYS527a/b is shown as SEQ ID NO: 63, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 64 is shown; the nucleotide sequence of the upstream primer for amplifying DYS533 is shown as SEQ ID NO: 65, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 66 is shown; the nucleotide sequence of the upstream primer for amplifying DYS444 is shown as SEQ ID NO: 67, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: as shown at 68.
Preferably, the 37Y-STR loci are divided into five groups, wherein,
the first group includes: DYS438, DYS389I, DYS448, DYS389II, DYS19, Y _ GATA _ H4, and DYS 518;
the second group includes: DYS460, DYS458, DYS437, DYS439, DYS643, DYF387S1, and DYS 385;
the third group includes: DYS393, DYS391, DYS390, DYS635, DYS449, and DYS 596;
the fourth group includes: DYS456, DYS576, DYS549, DYS570, DYS481, DYS392, and DYS 627;
the fifth group includes: y-indel, DYS388, DYS447, DYS557, DYS527, DYS533, and DYS 444;
the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.
The invention also provides a kit comprising the primer group in the scheme.
Preferably, the kit further comprises a PCR reaction premix and deionized water.
Preferably, the kit further comprises an internal molecular weight standard and an allelic ladder.
Preferably, in the primer set,
SEQ ID NO: 1 and SEQ ID NO: 2 is independently 0.284-0.37 mu M;
SEQ ID NO: 3 and SEQ ID NO: 4, the concentration of the primers is independently 0.017-0.03 mu M;
SEQ ID NO: 5 and SEQ ID NO: 6 is 0.22-0.28 mu M independently;
SEQ ID NO: 7 and SEQ ID NO: the concentration of the primers shown in 8 is 0.232-0.285 mu M independently;
SEQ ID NO: 9 and SEQ ID NO: 10 is 0.3-0.392 μ M independently;
SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.289-0.36 mu M;
SEQ ID NO: 13 and SEQ ID NO: the concentration of the primer shown in 14 is 0.21-0.3 mu M independently;
SEQ ID NO: 15 and SEQ ID NO: 16 is 0.048-0.13 mu M independently;
SEQ ID NO: 17 and SEQ ID NO: 18 is 0.052-0.12 mu M independently;
SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.038-0.12 mu M;
SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.082-0.169 mu M;
SEQ ID NO: 23 and SEQ ID NO: the concentration of the primers shown in 24 is 0.072-0.175 mu M independently;
SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.1-0.19 mu M;
SEQ ID NO: 27 and SEQ ID NO: 28 is independently 0.085-0.145 mu M;
SEQ ID NO: 29 and SEQ ID NO: 30 is independently 0.034-0.118 mu M;
SEQ ID NO: 31 and SEQ ID NO: 32 is independently 0.085-0.168 mu M;
SEQ ID NO: 33 and SEQ ID NO: 34 is independently 0.06-0.15 mu M;
SEQ ID NO: 35 and SEQ ID NO: 36 is independently 0.16-0.22 μ M;
SEQ ID NO: 37 and SEQ ID NO: 38 is 0.07-0.128 mu M independently;
SEQ ID NO: 39 and SEQ ID NO: 40 is 0.199-0.293 mu M independently;
SEQ ID NO: 41 and SEQ ID NO: 42 is independently 0.08-0.13 mu M;
SEQ ID NO: 43 and SEQ ID NO: 44 is independently 0.05-0.14 mu M;
SEQ ID NO: 45 and SEQ ID NO: 46 is independently 0.09-0.154 mu M;
SEQ ID NO: 47 and SEQ ID NO: 48 is 0.06-0.11 mu M independently;
SEQ ID NO: 49 and SEQ ID NO: the concentration of the primers shown by 50 is 0.19-0.252 mu M independently;
SEQ ID NO: 51 and SEQ ID NO: 52 is independently 0.42-0.5 mu M;
SEQ ID NO: 53 and SEQ ID NO: 54 is independently 0.267-0.32 mu M;
SEQ ID NO: 55 and SEQ ID NO: 56 is independently 0.052-0.12 mu M;
SEQ ID NO: 57 and SEQ ID NO: the concentration of the primer shown as 58 is 0.078-0.12 mu M independently;
SEQ ID NO: 59 and SEQ ID NO: the concentration of the primers shown as 60 is 0.08-0.133 mu M independently;
SEQ ID NO: 61 and SEQ ID NO: the concentration of the primers shown in 62 is 0.11-0.2 mu M independently;
SEQ ID NO: 63 and SEQ ID NO: the concentration of the primers shown by 64 is 0.074-0.141 mu M independently;
SEQ ID NO: 65 and SEQ ID NO: 66 is independently 0.277-0.345 MuM;
SEQ ID NO: 67 and SEQ ID NO: 68 is independently 0.18-0.26. mu.M.
Preferably, the PCR reaction premix takes deionized water as a solvent and comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer solution, 100-20KCL of 0mM, MgCl of 3.75-7.5 mM250 to 100mM (NH)4)2SO40.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction.
Preferably, the allelic ladder and the Y chromosome STR locus correspond in relationship to one another:
in the first group: DYS438 corresponds to 9,10,11,12,13, 14; DYS389I corresponds to 10,11,12,13,14, 15; DYS448 corresponds to 16,17,18,19,20,21,22,23, 24; DYS389II corresponds to 24,25,26,27,28,29,30,31,32,33, 34; DYS19 corresponds to 10,11,12,13,14,15,16,17,18,19, 20; y _ GATA _ H4 corresponds to 8,9,10,11,12, 13; DYS 518: 32,33,34,36,37,38,39, 41;
in the second group: DYS460 corresponds to 8,9,10,11,12,13, 14; DYS458 corresponds to 14,15,16,17,18,18.2,19,20,21, 22; DYS437 corresponds to 13,14,15, 16; DYS439 corresponds to 9,10,11,12,13,14,15,; DYS643 corresponds to 8,9,10,11,12,13,14, 16, 17; DYF387S1 corresponds to 33,34,35,36,37,38,39, 40; DYS385 corresponds to 9,10,11,12,13,14,15,16,17,19,20,21,22, 24;
in the third group: DYS393 corresponds to 8,9,10,11,12,13,14, 15, 16; DYS391 corresponds to 5,6,7,8,9,10,11,13,14,15, 16; DYS390 corresponds to 17,18,19,20,21,22,23,24,25,26, 27; DYS635 corresponds to 17,18,19,20,21,22,23,24,25, 26; DYS449 corresponds to 22,24,25,27,30,31,32,33,34,36,37,38,40,41, 42; DYS596 corresponds to 14,15, 16;
in the fourth group: DYS456 corresponds to 13,14,15,16,17, 18; DYS576 corresponds to 11,12,13,14,15,16, 17; DYS549 corresponds to 7,8,9,10,12,15,16, 17; DYS570 corresponds to 10,11,12,13,15,16,17,18,19,20,21,23,24, 25; DYS481 corresponds to 16,17,18,20,21,22,26,27,28,29,30,31, 32; DYS392 corresponds to 7,9,10,11,12,13,14,15, 16; DYS627 corresponds to 16,17,18,19,20,21,22,23,24, 25;
in the fifth group: y-indel corresponds to 1, 2; DYS388 corresponds to 10,11,12,13,14, 15; DYS447 corresponds to 22,23,24,25,26,27,28,29, 30; DYS557 corresponds to 14,15,16,18,19,20, 21; DYS527 corresponds to 13,14,16,17,18,20,21,22,23,24,25,26, 27; DYS533 corresponds to 7,8,9,10,11,12,14,15, 16; DYS444 corresponds to 10,11,12,13,14,15, 16.
The invention also provides application of the primer group and the kit in the scheme in forensic individual identification, forensic DNA database construction, suspect family investigation or judicial genetic relationship identification.
Preferably, the application comprises the following steps:
putting a sample to be detected into an amplification system containing the primer group in the scheme, performing PCR amplification to obtain an amplification product, and detecting the amplification product;
when the sample to be detected is an original human body sample, the amplification system of the PCR amplification is calculated by 10 mu L and comprises: 2. mu.l of the primer set mixture, 4. mu.l of the PCR reaction premix and 4. mu.l of deionized water;
when the sample to be detected is human genome DNA, the amplification system of PCR amplification is counted by 10 mu L and comprises: 1. mu.l of human genomic DNA, 2. mu.l of the mixture of the primer sets, 4. mu.l of the PCR reaction premix and 3. mu.l of deionized water;
the amplification procedure of the PCR amplification is as follows: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃,40 sec, 72 ℃, 45sec, 30 cycles; 60 deg.C, 30 min.
The invention has the beneficial effects that: the invention provides a primer group for simultaneously amplifying 37Y-STR loci of a human, wherein the 37Y-STR loci comprise 36Y chromosome STR loci and 1Y-indel locus; the 37Y-STR loci have certain compatibility with the loci of the market mainstream kit, and can share and exchange with the existing DNA data. The 37Y-STR loci comprise 20 core loci specified by the ministry of public security, 15 preferred loci, all locus loci of the mainstream kit in the market at present, and 1Y-indel locus. The 37 gene loci are combined to provide more information, have the characteristics of high individual identification power and high non-paternity exclusion rate, and can be applied to the research fields of forensic paternity testing, individual identification, anthropology and the like. The primer group disclosed by the invention can realize rapid amplification, has strong amplification specificity and no non-specific amplification, and simultaneously, the primers do not interfere with each other and do not form primer dimers. The length of each primer is 18-32 bp, the TM value is about 60 ℃, each pair of primers has high specificity, and all 37 pairs of primers have no interaction, so that the primers corresponding to 37 loci can be compatible with one single tube. The length of the overall amplification product is between 70 and 530 bp. After amplification reaction, 1-2 specific amplification bands can be obtained from each locus, and non-specific amplification peaks, primer peaks and other miscellaneous peaks are avoided. The primer group also has the advantage of high sensitivity, can be suitable for the amplification detection of the autosomal STR of various test materials, and can meet the requirements of multifunctional STR identification of the current forensic identity identification, the construction of a forensic DNA database and the identification of judicial genetic relationship.
Drawings
FIG. 1 is a positive control DNA9948 genotyping map;
FIG. 2 is a ladder allelic typing map;
FIG. 3 is a BTY-550 spectrum of a molecular weight internal standard;
FIG. 4 is a chart of suspected subgenomic typing;
FIG. 5 is a suspected father genotyping map;
FIG. 6 is a blood card test material genotyping chart;
FIG. 7 is a genotyping chart of the extracted DNA sample.
Detailed Description
The invention provides a primer group for simultaneously amplifying 37Y-STR loci of a human, wherein the 37Y-STR loci comprise 36Y chromosome STR loci and 1Y-indel locus; the 36Y chromosome STR loci are respectively: DYS438, DYS389I, DYS448, DYS389II, DYS19, Y _ GATA _ H4, DYS518, DYS460, DYS458, DYS437, DYS439, DYS643, dysf 387S1a/b, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS449, DYS596, DYS456, DYS576, DYS549, DYS570, DYS481, DYS392, DYS627, DYS388, DYS447, DYS557, DYS527a/b, DYS533 and DYS 444;
the nucleotide sequence of the upstream primer for amplifying DYS438 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS389I is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS389II is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS448 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS19 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying Y _ GATA _ H4 is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification; the nucleotide sequence of the upstream primer for amplifying DYS518 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS460 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS458 is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS437 is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS439 is shown as SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22; the nucleotide sequence of the upstream primer for amplifying DYS643 is shown as SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24; the nucleotide sequence of the upstream primer for amplifying DYF387S1a/b is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 26 is shown; the nucleotide sequence of the upstream primer for amplifying DYS385a/b is shown as SEQ ID NO: 27, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 28 is shown; the nucleotide sequence of the upstream primer for amplifying DYS393 is shown in SEQ ID NO: 29, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 30 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS391 is shown as SEQ ID NO: 31, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 32 is shown; the nucleotide sequence of the upstream primer for amplifying DYS390 is shown as SEQ ID NO: 33, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 34; the nucleotide sequence of the upstream primer for amplifying DYS635 is shown as SEQ ID NO: 35, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 36 is shown; the nucleotide sequence of the upstream primer for amplifying DYS449 is shown as SEQ ID NO: 37, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 38; the nucleotide sequence of the upstream primer for amplifying DYS596 is shown as SEQ ID NO: 39, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 40 is shown in the figure; the nucleotide sequence of the upstream primer for amplifying DYS456 is shown as SEQ ID NO: 41, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 42 is shown; the nucleotide sequence of the upstream primer for amplifying DYS576 is shown as SEQ ID NO: 43, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 44 is shown; the nucleotide sequence of the upstream primer for amplifying DYS549 is shown as SEQ ID NO: 45, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 46; the nucleotide sequence of the upstream primer for amplifying DYS570 is shown as SEQ ID NO: 47, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 48 is shown; the nucleotide sequence of the upstream primer for amplifying DYS481 is shown as SEQ ID NO: 49, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 50 is shown; the nucleotide sequence of the upstream primer for amplifying DYS392 is shown as SEQ ID NO: 51, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 52; the nucleotide sequence of the upstream primer for amplifying DYS627 is shown as SEQ ID NO: 53, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 54 is shown; the nucleotide sequence of the upstream primer for amplifying the Y-indel is shown as SEQ ID NO: 55, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 56 is shown; the nucleotide sequence of the upstream primer for amplifying DYS388 is shown as SEQ ID NO: 57, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 58; the nucleotide sequence of the upstream primer for amplifying DYS447 is shown as SEQ ID NO: 59, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 60 is shown; the nucleotide sequence of the upstream primer for amplifying DYS557 is shown as SEQ ID NO: 61, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 62; the nucleotide sequence of the upstream primer for amplifying DYS527a/b is shown as SEQ ID NO: 63, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 64 is shown; the nucleotide sequence of the upstream primer for amplifying DYS533 is shown as SEQ ID NO: 65, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 66 is shown; the nucleotide sequence of the upstream primer for amplifying DYS444 is shown as SEQ ID NO: 67, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: as shown at 68.
The 37 loci tested by the invention comprise: the 36Y chromosome STR loci are: DYS438, DYS389I, DYS448, DYS389II, DYS19, Y _ GATA _ H4, DYS518, DYS460, DYS458, DYS437, DYS439, DYS643, dysf 387S1a/b, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS449, DYS596, DYS456, DYS576, DYS549, DYS570, DYS481, DYS392, DYS627, DYS388, DYS447, DYS557, DYS527a/b, DYS533, DYS444 and 1Y-indel locus.
The primer set of the present invention comprises 37 pairs of primers, each corresponding to 37 loci involved in the present invention. And (2) designing 37 pairs of primers aiming at 37 loci, wherein the length of the primers is between 18bp and 30bp, the TM value is about 60 ℃, each pair of primers has high specificity, and all 37 pairs of primers have no interaction, so that the primers corresponding to 37 loci can be compatible in a single tube. The length of the total amplification product is between 70 and 520 bp. After amplification reaction, 1-2 specific amplification bands can be obtained from each locus, and non-specific amplification peaks, primer peaks and other miscellaneous peaks are avoided.
The 37Y-STR loci comprise 20 core loci specified by the ministry of public security, 15 preferred loci, all locus loci of the mainstream kit in the market at present, and 1Y-indel locus. The 37 gene loci are combined to provide more information, have the characteristics of high individual identification power and high non-paternity exclusion rate, and can be applied to the research fields of forensic paternity testing, individual identification, anthropology and the like. The Y-STR locus in the primer set of the invention is compared with the sites of the mainstream kit in the market, and the comparison is shown in Table 1.
Table 1 comparison of the Y-STR locus in the primer set of the invention with the commercial mainstream kit sites is shown in table 1.
| Baite Yuan | ABI | Promega | |
| The invention | YFiler27 | PP23 | |
| DYS438 | + | + | + |
| DYS389I | + | + | + |
| DYS448 | + | + | + |
| DYS389II | + | + | + |
| DYS19 | + | + | + |
| YGATAH4 | + | + | + |
| DYS518 | + | + | |
| DYS460 | + | + | |
| DYS458 | + | + | + |
| DYS437 | + | + | + |
| DYS439 | + | + | + |
| DYS643 | + | + | |
| DYF387S1a/b | + | + | |
| DYS385a/b | + | + | + |
| DYS393 | + | + | + |
| DYS391 | + | + | + |
| DYS390 | + | + | + |
| DYS635 | + | + | + |
| DYS449 | + | + | |
| DYS596 | + | ||
| DYS456 | + | + | + |
| DYS576 | + | + | + |
| DYS549 | + | + | |
| DYS570 | + | + | + |
| DYS481 | + | + | + |
| DYS392 | + | + | + |
| DYS627 | + | + | |
| Y-indel | + | ||
| DYS388 | + | ||
| DYS447 | + | ||
| DYS557 | + | ||
| DYS527 | + | ||
| DYS533 | + | + | + |
| DYS444 | + | + |
In the invention, the amplification balance of the primer group comprehensively exceeds the standard (more than or equal to 70% in a gene locus, more than or equal to 50% in the same color group, and more than or equal to 30% among different color groups) in GB/T37226-2018 court science human fluorescence labeling STR composite amplification detection reagent quality basic requirements.
In the present invention, the 37Y-STR loci are preferably divided into five groups, wherein the first group comprises: DYS438, DYS389I, DYS448, DYS389II, DYS19, Y _ GATA _ H4, and DYS 518; the second group includes: DYS460, DYS458, DYS437, DYS439, DYS643, DYF387S1, and DYS 385; the third group includes: DYS393, DYS391, DYS390, DYS635, DYS449, and DYS 596; the fourth group includes: DYS456, DYS576, DYS549, DYS570, DYS481, DYS392, and DYS 627; the fifth group includes: y-indel, DYS388, DYS447, DYS557, DYS527, DYS533, and DYS 444; the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.
In the present invention, the fluorescent dye used in the first group is preferably FAM; the fluorescent dye adopted by the second group is preferably HEX; the fluorescent dye used in the third group is preferably TAMAR; the fluorescent dye adopted by the fourth group is preferably ROX; the fluorescent dye adopted in the fifth group is preferably PURPLE; ORG was used as internal standard. The invention adopts six-color fluorescent labeling systems, namely FAM, HEX, TAMAR, ROX, PURPLE and ORG. Wherein FAM represents blue, HEX represents green, TAMAR represents yellow, ROX represents red, PURPLE represents PURPLE, and ORG represents orange. The invention can simultaneously amplify 37 gene loci in one reaction, and fully meets the compatibility of the current public security DNA database comparison.
In the present invention, the 5' end of the upstream primer and/or the downstream primer of each of the Y-STR loci is labeled with a fluorescent dye. The invention uses the fluorescence labeling method to label a fluorescent dye at the 5' end of the primer, the PCR product can emit optical signals with specific wavelength under the laser excitation state, the optical signals can be collected by electrophoresis detection through a genetic analyzer (ABI 3130/ABI 3500/ABI 3730 series one by one), and the detection is carried out through the collected optical signals.
The invention also provides a kit comprising the primer group in the scheme.
In the present invention, the kit preferably further comprises a PCR reaction premix and deionized water. In the present invention, the kit preferably further comprises a positive control DNA 9948. In the invention, the primer group, the PCR reaction premixed solution and the deionized water form a reaction system of the kit.
In the present invention, in the primer set, SEQ ID NO: 1 and SEQ ID NO: 2 is independently preferably 0.284-0.37 mu M, and is further preferably 0.329 mu M; SEQ ID NO: 3 and SEQ ID NO: 4, the concentration of the primer is preferably 0.017-0.03 mu M independently, and more preferably 0.022 mu M; SEQ ID NO: 5 and SEQ ID NO: 6 is independently preferably 0.22-0.28 mu M, and more preferably 0.25 mu M; SEQ ID NO: 7 and SEQ ID NO: the concentration of the primer shown in 8 is preferably 0.232-0.285 mu M independently, and more preferably 0.246 mu M independently; SEQ ID NO: 9 and SEQ ID NO: 10 is preferably 0.3-0.392 μ M, and more preferably 0.349 μ M; SEQ ID NO: 11 and SEQ ID NO: 12 is independently preferably 0.289-0.36 mu M, and more preferably 0.329 mu M; SEQ ID NO: 13 and SEQ ID NO: the concentration of the primer shown in 14 is 0.21-0.3 mu M independently and preferably 0.258 mu M; SEQ ID NO: 15 and SEQ ID NO: 16 is preferably 0.048-0.13 mu M independently, and is further preferably 0.074 mu M; SEQ ID NO: 17 and SEQ ID NO: 18 independently, the concentration of the primer is preferably 0.052-0.12. mu.M, and more preferably 0.092. mu.M; SEQ ID NO: 19 and SEQ ID NO: 20 is independently preferably 0.038-0.12 mu M, and further preferably 0.082 mu M; SEQ ID NO: 21 and SEQ ID NO: 22 is independently preferably 0.082-0.169 mu M, and is further preferably 0.126 mu M; SEQ ID NO: 23 and SEQ ID NO: 24 is preferably 0.072-0.175 mu M independently, and more preferably 0.13 mu M; SEQ ID NO: 25 and SEQ ID NO: 26 is independently preferably 0.1-0.19. mu.M, and more preferably 0.13. mu.M; SEQ ID NO: 27 and SEQ ID NO: 28, the concentration of the primer is preferably 0.085-0.145 mu M independently, and more preferably 0.117 mu M; SEQ ID NO: 29 and SEQ ID NO: 30 is preferably 0.034-0.118 muM, and more preferably 0.072 muM; SEQ ID NO: 31 and SEQ ID NO: 32 is preferably 0.085-0.168 mu M independently, and is further preferably 0.125 mu M independently; SEQ ID NO: 33 and SEQ ID NO: 34 is independently preferably 0.06-0.15 mu M, and further preferably 0.105 mu M; SEQ ID NO: 35 and SEQ ID NO: the concentration of the primer shown in 36 is preferably 0.16-0.22 mu M independently, and more preferably 0.185 mu M; SEQ ID NO: 37 and SEQ ID NO: 38 is preferably 0.07-0.128. mu.M, and more preferably 0.101. mu.M; SEQ ID NO: 39 and SEQ ID NO: 40 is independently preferably 0.199-0.293 mu M, and further preferably 0.251 mu M; SEQ ID NO: 41 and SEQ ID NO: 42 is independently preferably 0.08-0.13 mu M, and more preferably 0.1 mu M; SEQ ID NO: 43 and SEQ ID NO: 44 is independently preferably 0.05-0.14 mu M, and further preferably 0.073 mu M; SEQ ID NO: 45 and SEQ ID NO: 46 is independently preferably 0.09-0.154 mu M, and further preferably 0.135 mu M; SEQ ID NO: 47 and SEQ ID NO: 48 is independently preferably 0.06-0.11 mu M, and further preferably 0.085 mu M; SEQ ID NO: 49 and SEQ ID NO: 50 is independently preferably 0.19-0.252 mu M, and more preferably 0.226 mu M; SEQ ID NO: 51 and SEQ ID NO: 52 is independently preferably 0.42-0.5 mu M, and more preferably 0.467 mu M; SEQ ID NO: 53 and SEQ ID NO: 54 is independently preferably 0.267-0.32 mu M, and further preferably 0.299 mu M; SEQ ID NO: 55 and SEQ ID NO: 56 is preferably 0.052 to 0.12. mu.M, more preferably 0.082. mu.M; SEQ ID NO: 57 and SEQ ID NO: the concentration of the primer shown by 58 is preferably 0.078-0.12 mu M independently, and more preferably 0.092 mu M; SEQ ID NO: 59 and SEQ ID NO: the concentration of the primer shown as 60 is preferably 0.08-0.133 mu M independently, and more preferably 0.102 mu M; SEQ ID NO: 61 and SEQ ID NO: 62 is preferably 0.11-0.2. mu.M, and more preferably 0.158. mu.M; SEQ ID NO: 63 and SEQ ID NO: 64 is independently preferably 0.074-0.141. mu.M, and more preferably 0.102. mu.M; SEQ ID NO: 65 and SEQ ID NO: 66 is independently preferably 0.277 to 0.345 mu M, and further preferably 0.307 mu M; SEQ ID NO: 67 and SEQ ID NO: 68 is independently preferably 0.18 to 0.26. mu.M, and more preferably 0.24. mu.M.
In the present invention, the PCR reaction premix preferably includes hot-start DNA polymerase, dNTP, magnesium ions, potassium ions, Tris buffer and enhancer; the PCR reaction premix takes deionized water as a solvent, and preferably comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl250 to 100mM (NH)4)2SO40.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction. In one embodiment of the present invention, the recipe for the PCR reaction premix is shown in Table 2.
TABLE 2 PCR reaction premix formula of the inventive example
H in Table 22O is deionized water which is highly deionized water, the resistivity reaches 18.2M omega, and the interference of external ions on an amplification system is completely eliminated.
In the invention, the PCR reaction premix has the characteristics of high speed, high sensitivity, strong adaptability and the like, and can not freeze at the temperature of-20 ℃, thereby effectively preventing the influence of freeze thawing on the performance of the reagent. The sensitivity of the PCR reaction premix liquid is as high as 0.03ng DNA, the effective amplification time is reduced to be within 1.5h, the PCR reaction premix liquid has excellent amplification effect on DNA from different sources (blood, blood stains, semen, seminal stains, saliva stains, hair, tissues, nails, body fluid and the like), and meanwhile, the PCR reaction premix liquid also has very good amplification effect on special blood cards, saliva cards and FTA cards.
In the invention, the positive control DNA9948 is used as an amplification standard substance for testing the quality of an amplification system. In the preferred embodiment of the invention, the positive control can be correctly typed, and the sensitivity is as high as 0.03ng and is far lower than the standard 0.125 ng. The 9948 fractal pattern amplified by the present invention is shown in FIG. 1 and Table 3.
TABLE 39948 genotyping
In the present invention, the kit preferably further comprises an internal molecular weight standard and an allelic ladder. In the present invention, the molecular weight internal standard and the allele ladder are detection reagents of the kit.
In the present invention, the correspondence between the allelic ladder and the Y chromosome STR locus is specifically found in table 4:
TABLE 4 correspondence of allelic ladders and the Y chromosome STR loci
In the present invention, the allele ladder encompasses all the most common alleles and most rare alleles at each locus, which is very convenient for data analysis and comparison of genotyping results.
In the present invention, the molecular weight internal standard is BTY-550; the BTY-550 comprises the following DNA fragments: 65. 75, 100, 139, 150, 160, 200, 250, 300, 340, 400, 450, 490, 500, 540, and 550. In the present invention, the internal molecular weight standard is labeled with the fluorescent dye ORG, representing orange. The molecular weight internal standard can effectively distinguish the sizes of 65-550 bp DNA fragments.
In the specific implementation process of the invention, the kit comprises two parts, wherein the first part is a reaction system and comprises a primer group, a reaction premix, a positive control DNA9948 and deionized water; the second part is a detection reagent comprising a molecular weight internal standard and an allele ladder.
The invention also provides application of the primer group and the kit in the scheme in forensic individual identification, forensic DNA database construction, suspect family investigation or judicial genetic relationship identification.
In the present invention, the application preferably comprises the steps of:
putting a sample to be detected into an amplification system containing the primer group in the scheme, performing PCR amplification to obtain an amplification product, and detecting the amplification product;
when the sample to be detected is an original human body sample, the amplification system of the PCR amplification is calculated by 10 mu L and comprises: 2. mu.l of the primer set mixture, 4. mu.l of the PCR reaction premix and 4. mu.l of deionized water;
when the sample to be detected is human genome DNA, the amplification system of PCR amplification is counted by 10 mu L and comprises: 1. mu.l of human genomic DNA, 2. mu.l of the mixture of the primer sets, 4. mu.l of the PCR reaction premix and 3. mu.l of deionized water;
the amplification procedure of the PCR amplification is as follows: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃,40 sec, 72 ℃, 45sec, 30 cycles; 60 deg.C, 30 min.
In the present invention, the sample to be detected preferably includes human blood, blood stain, semen, seminal stain, saliva, salivary stain, hair, tissue, nail or body fluid.
In the present invention, the standard amplification system for PCR amplification is 25. mu.l, and comprises 5. mu.l of the mixture of the primer sets, 10. mu.l of the PCR reaction premix, 7.5. mu.l of deionized water, and 2.5. mu.l of the positive control DNA 9948. The amplification procedure for the standard amplification system is shown in Table 5.
TABLE 5 amplification procedure for the Standard amplification System
The method for detecting the amplification product of the present invention preferably includes: carrying out electrophoretic detection on the amplification product; the electrophoresis detection equipment is preferably ABI3500 XL; in the specific implementation process of the invention, HiDi formamide, molecular weight internal standard BTY-500 and PCR amplification product or allele ladder 27A are mixed to obtain a mixture; taking the volume of HiDi formamide as 8.5 μ l, and measuring the molecular weight internal standard BTY-5000.5 μ l, PCR amplification product or allele step 27A 1 μ l; after the mixture was dispensed into a 96-well plate, centrifugation was carried out to remove air bubbles, and the 96-well plate was placed in a sample application tray and then in a 3500Xl genetic analyzer at a sample application voltage of 1.8kVolts for 12sec, after which electrophoresis was started.
The present invention preferably further comprises analyzing the results of the electrophoresis on GeneMapper _ IDX software after said electrophoresis. The positive control 9948 typing map is shown in FIG. 1, and the allelic ladder map is shown in FIG. 2; molecular weight internal standard BTY-550 is shown in FIG. 3.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Two hairs from the suspected father and son were subjected to direct multiplex amplification, and 37 loci were detected. The amplification method adopts a direct amplification method, namely a section of hair with hair follicles is taken and directly put into an amplification system, so that the hair follicles are completely immersed in liquid, the standard amplification program is adopted in the amplification program, an ABI Proflex PCR instrument is adopted in an amplification instrument, a 3500xl genetic analyzer is adopted in a genetic analyzer, and GeneMapper-ID-X analysis software is adopted in analysis software. The operation steps of this embodiment are as follows:
first, 0.5cm above the hair follicles was cut short with scissors, and the cut hair with follicles was placed in a 200. mu.l PCR tube.
Preparing 10 mul amplification system according to the standard reaction system of the invention: mu.l of the primer mixture, 4. mu.l of the reaction premix, and 4. mu.l of deionized water were added to the PCR tube with cut hair. Performing PCR amplification on an ABI Proflex PCR instrument by the following amplification program: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃,40 sec, 72 ℃, 45sec, 30 cycles; 60 ℃ for 30 min; storing at constant temperature of 10 ℃.
Detecting and data analyzing by genetic analyzer
After PCR amplification is finished, the amplification product is detected on ABI3500 XL. 8.5. mu.l of HiDi formamide + 0.5. mu.l of molecular weight internal standard BTY-500+ 1. mu.l of PCR product/1. mu.l of allele step 27A; after being divided into 96-well plates, the plates were centrifuged to remove air bubbles, and the 96-well plates were placed in a sample tray and then in a 3500Xl genetic analyzer at a sample injection voltage of 1.8kVolts for 12sec, after which electrophoresis was started.
Analysis was performed on GeneMapper IDX software after the end of electrophoresis, see fig. 4: a suspected gene fractal graph; FIG. 5: a suspected father genotyping map; the results of the suspected parent and child gene typing are shown in Table 6.
Table 6: suspected paternal genotyping
By analyzing and comparing the obtained genotypes of the 37 loci, the invention can find that the gene information of the 37 loci of the suspected father and son conforms to the genetic rule, and can judge the father-son relationship.
Example 2: application of kit of the invention in forensic medicine
The invention is mainly used for the construction of DNA database of criminals and the identification of the identity of criminal suspects in the field of forensic science.
The national forensic DNA database is a national DNA database of illegal criminals established by the ministry of public security in China, breaks through 5000 thousands of data in the database at present, and is a main tool for case detection of a public security system. The database is usually built by adopting a method of directly amplifying and detecting blood samples, the detection material in the identification work of the identity of the criminal suspect is usually a complex detection material, and the amplification detection method is adopted for detection after DNA is extracted. The invention can take two different inspection materials into consideration, and is convenient for the construction of DNA databases in China and the identification of the identity of the criminal suspect. The detailed part of the operation is as follows:
firstly, it is known that the material to be detected by the criminal is usually blood card, which can be directly amplified. Using a 1mm diameter punch, a 1mm diameter blood piece was punched directly from the dried blood card and placed in a 200. mu.l PCR tube.
Secondly, unknown criminals are generally case field inspection materials which are complex, and the detection analysis of data is generally carried out by a method of firstly extracting DNA and then amplifying. DNA was extracted by the Chelex-100 and magnetic bead method. (the extraction method refers to DNA extraction of Zhengxiufen 'forensic DNA analysis' Chapter four).
Thirdly, the preparation and amplification of the reaction system, referring to the standard system of the invention, 10 mul of reaction system is prepared, as shown in Table 7 below. Performing PCR amplification on an ABI Proflex PCR instrument by the following amplification program: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃,40 sec, 72 ℃, 45sec, 30 cycles; 60 ℃ for 30 min; storing at constant temperature of 10 ℃.
Table 7: reaction System of this example
Fourthly, detecting by a genetic analyzer and analyzing data
After PCR amplification is finished, the amplification product is detected on ABI3500 xl. 8.5. mu.l of HiDi formamide + 0.5. mu.l of molecular weight internal standard BTY-500+ 1. mu.l of PCR product/1. mu.l of allele step 44Y; after being divided into 96-well plates, the plates were centrifuged to remove air bubbles, and the 96-well plates were placed in a sample tray and then in a 3500Xl genetic analyzer at a sample injection voltage of 1.8kVolts for 12sec, after which electrophoresis was started.
And after the electrophoresis is finished, analyzing on GeneMapper _ IDX software, exporting the analyzed data to a CODIS format file after the analysis is finished, and uploading the exported data to a national DNA database. FIG. 6 is a blood card sample typing chart, and FIG. 7 is a DNA extraction sample typing chart. As can be seen from FIGS. 6 and 7, the invention detects the direct amplification sample and the extracted DNA sample, has correct result and good graphic balance, and can completely meet the daily application of the forensic.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Baite Yuan Biotechnology (Beijing) Ltd
<120> primer group and kit for simultaneously amplifying 37 human Y-STR loci and application thereof
<160> 68
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
caaaattagt ggggaatagt t 21
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctgtccctga gtagcagaag aa 22
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtctggaaat ttatctctat ctt 23
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cactatgact actgagtttc tgt 23
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctgaagagct aaacagagac ctaag 25
<210> 6
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggcaacacaa gtgaaactgc ttctcg 26
<210> 7
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gtctggaaat ttatctctat ctt 23
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gaggaatctg acacctctga ca 22
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gcaacaggaa tgaaactcca a 21
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tcctgaatgg tacttcctag gttt 24
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ctggttaaac tactgtgcct tt 22
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gccacagtgt gagaagtgtg a 21
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tgtcatgaac tgaaatgatg gc 22
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gtggtcttct acttgtgtca atac 24
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ctattcattc aatcatacac cc 22
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tatattttac acatttttgg gc 22
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
agtgtctcac ttcaagcacc aa 22
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
<210> 20
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tggttcacag aaataaagaa ttccatg 27
<210> 21
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ggaccttgtg ataatgtaag atagatag 28
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
<210> 23
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
caattaggta ggtaaagagg aagatg 26
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acagctcacc agaaggttgc aagac 25
<210> 26
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
actcttggca tctagtgact ctcatatc 28
<210> 27
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
actggagaat tgcttggacc caggatgg 28
<210> 28
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ctgtctttga gcaagaaaaa tagtaccc 28
<210> 29
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ttcatgtgag ttagccgttt agc 23
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
<210> 31
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ttaatctgat ttctgaacct actg 24
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
aacatctttg tcatctacct aat 23
<210> 34
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ctctcttctc cactttaacc agtatacag 29
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
aagagtgaaa ctccatttca aat 23
<210> 36
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gcaactctca tctgtattat ctatgta 27
<210> 37
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
cagacagaaa gggagataga g 21
<210> 38
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gcaactctca tctgtattat ctat 24
<210> 39
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tctaatatgc caccctttta ttatttctac 30
<210> 40
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
ggtgaagtaa tggaattaga aat 23
<210> 41
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ggtttctgtt caagtcttga gtct 24
<210> 42
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
atccatatca tctatcctct gcct 24
<210> 43
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tcccaaagtt ctggcattac aa 22
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gaggaagacc ctgtcattca c 21
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
<210> 46
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gcataggaat gacacaataa ac 22
<210> 47
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
tactctggag gctgaggcac gataatga 28
<210> 48
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ttattgggat gctaggtaaa gct 23
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gaactcaagt ccaaaaaatg agg 23
<210> 50
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
<210> 51
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
<210> 52
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gatacattgt ttatagcagc aaaa 24
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tggagtttgc tgtaagctaa gat 23
<210> 54
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
cacatataca aaacattgat ttttgtttg 29
<210> 55
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
ctagagggac agaactaatg gaatatc 27
<210> 56
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
aggagatggg agtaataagc gtat 24
<210> 57
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
gcatagaggt gttcagaata gtc 23
<210> 58
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tcagcatagt caagaaacca gac 23
<210> 59
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tgtgttttct ggggttgctt tctg 24
<210> 60
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gagaagtcaa aacagaggga tc 22
<210> 61
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
accatgtgga taatgagcaa atgg 24
<210> 62
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tgataccttt gtttctgttc attcttg 27
<210> 63
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gcggagcttt tagtgagcca ga 22
<210> 64
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
gcttgctttg cgttatctct gc 22
<210> 65
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
gtttgaccca gtgatatgtt ct 22
<210> 66
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
<210> 67
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
taatctattc agattcagct tttgg 25
<210> 68
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ctgtattatt acaataaagt aacctagc 28
Claims (10)
1. A primer group for simultaneously amplifying 37Y-STR loci of a human, wherein the 37Y-STR loci comprise 36Y chromosome STR loci and 1Y-indel locus; the 36Y chromosome STR loci are respectively: DYS438, DYS389I, DYS448, DYS389II, DYS19, Y _ GATA _ H4, DYS518, DYS460, DYS458, DYS437, DYS439, DYS643, dysf 387S1a/b, DYS385a/b, DYS393, DYS391, DYS390, DYS635, DYS449, DYS596, DYS456, DYS576, DYS549, DYS570, DYS481, DYS392, DYS627, DYS388, DYS447, DYS557, DYS527a/b, DYS533 and DYS 444;
the nucleotide sequence of the upstream primer for amplifying DYS438 is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying DYS389I is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 4 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying DYS389II is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying DYS448 is shown as SEQ ID NO: 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 8 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying DYS19 is shown as SEQ ID NO: 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 10 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying Y _ GATA _ H4 is shown as SEQ ID NO: 11, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 12 is shown in the specification;
the nucleotide sequence of the upstream primer for amplifying DYS518 is shown as SEQ ID NO: 13, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 14 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying DYS460 is shown as SEQ ID NO: 15, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 16 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying DYS458 is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying DYS437 is shown as SEQ ID NO: 19, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 20 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying DYS439 is shown as SEQ ID NO: 21, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 22;
the nucleotide sequence of the upstream primer for amplifying DYS643 is shown as SEQ ID NO: 23, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 24;
the nucleotide sequence of the upstream primer for amplifying DYF387S1a/b is shown as SEQ ID NO: 25, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 26 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS385a/b is shown as SEQ ID NO: 27, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 28 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS393 is shown in SEQ ID NO: 29, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 30 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying DYS391 is shown as SEQ ID NO: 31, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 32 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS390 is shown as SEQ ID NO: 33, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 34;
the nucleotide sequence of the upstream primer for amplifying DYS635 is shown as SEQ ID NO: 35, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 36 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS449 is shown as SEQ ID NO: 37, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 38;
the nucleotide sequence of the upstream primer for amplifying DYS596 is shown as SEQ ID NO: 39, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 40 is shown in the figure;
the nucleotide sequence of the upstream primer for amplifying DYS456 is shown as SEQ ID NO: 41, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 42 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS576 is shown as SEQ ID NO: 43, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 44 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS549 is shown as SEQ ID NO: 45, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 46;
the nucleotide sequence of the upstream primer for amplifying DYS570 is shown as SEQ ID NO: 47, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 48 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS481 is shown as SEQ ID NO: 49, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 50 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS392 is shown as SEQ ID NO: 51, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 52;
the nucleotide sequence of the upstream primer for amplifying DYS627 is shown as SEQ ID NO: 53, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 54 is shown;
the nucleotide sequence of the upstream primer for amplifying the Y-indel is shown as SEQ ID NO: 55, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 56 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS388 is shown as SEQ ID NO: 57, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 58;
the nucleotide sequence of the upstream primer for amplifying DYS447 is shown as SEQ ID NO: 59, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 60 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS557 is shown as SEQ ID NO: 61, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: shown at 62;
the nucleotide sequence of the upstream primer for amplifying DYS527a/b is shown as SEQ ID NO: 63, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 64 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS533 is shown as SEQ ID NO: 65, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 66 is shown;
the nucleotide sequence of the upstream primer for amplifying DYS444 is shown as SEQ ID NO: 67, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: as shown at 68.
2. The primer set of claim 1, wherein said 37Y-STR loci are grouped into five groups, wherein,
the first group includes: DYS438, DYS389I, DYS448, DYS389II, DYS19, Y _ GATA _ H4, and DYS 518;
the second group includes: DYS460, DYS458, DYS437, DYS439, DYS643, DYF387S1, and DYS 385;
the third group includes: DYS393, DYS391, DYS390, DYS635, DYS449, and DYS 596;
the fourth group includes: DYS456, DYS576, DYS549, DYS570, DYS481, DYS392, and DYS 627;
the fifth group includes: y-indel, DYS388, DYS447, DYS557, DYS527, DYS533, and DYS 444;
the primers of the first set to the fifth set of STR loci are different from each other in fluorescent dye.
3. A kit comprising the primer set of claim 1 or 2.
4. The kit of claim 3, further comprising a PCR reaction premix and deionized water.
5. The kit of claim 3 or 4, further comprising an internal molecular weight standard and an allelic ladder.
6. The kit according to claim 3, wherein in the primer set,
SEQ ID NO: 1 and SEQ ID NO: 2 is independently 0.284-0.37 mu M;
SEQ ID NO: 3 and SEQ ID NO: 4, the concentration of the primers is independently 0.017-0.03 mu M;
SEQ ID NO: 5 and SEQ ID NO: 6 is 0.22-0.28 mu M independently;
SEQ ID NO: 7 and SEQ ID NO: the concentration of the primers shown in 8 is 0.232-0.285 mu M independently;
SEQ ID NO: 9 and SEQ ID NO: 10 is 0.3-0.392 μ M independently;
SEQ ID NO: 11 and SEQ ID NO: 12 is independently 0.289-0.36 mu M;
SEQ ID NO: 13 and SEQ ID NO: the concentration of the primer shown in 14 is 0.21-0.3 mu M independently;
SEQ ID NO: 15 and SEQ ID NO: 16 is 0.048-0.13 mu M independently;
SEQ ID NO: 17 and SEQ ID NO: 18 is 0.052-0.12 mu M independently;
SEQ ID NO: 19 and SEQ ID NO: 20 is independently 0.038-0.12 mu M;
SEQ ID NO: 21 and SEQ ID NO: 22 is independently 0.082-0.169 mu M;
SEQ ID NO: 23 and SEQ ID NO: the concentration of the primers shown in 24 is 0.072-0.175 mu M independently;
SEQ ID NO: 25 and SEQ ID NO: 26 is independently 0.1-0.19 mu M;
SEQ ID NO: 27 and SEQ ID NO: 28 is independently 0.085-0.145 mu M;
SEQ ID NO: 29 and SEQ ID NO: 30 is independently 0.034-0.118 mu M;
SEQ ID NO: 31 and SEQ ID NO: 32 is independently 0.085-0.168 mu M;
SEQ ID NO: 33 and SEQ ID NO: 34 is independently 0.06-0.15 mu M;
SEQ ID NO: 35 and SEQ ID NO: 36 is independently 0.16-0.22 μ M;
SEQ ID NO: 37 and SEQ ID NO: 38 is 0.07-0.128 mu M independently;
SEQ ID NO: 39 and SEQ ID NO: 40 is 0.199-0.293 mu M independently;
SEQ ID NO: 41 and SEQ ID NO: 42 is independently 0.08-0.13 mu M;
SEQ ID NO: 43 and SEQ ID NO: 44 is independently 0.05-0.14 mu M;
SEQ ID NO: 45 and SEQ ID NO: 46 is independently 0.09-0.154 mu M;
SEQ ID NO: 47 and SEQ ID NO: 48 is 0.06-0.11 mu M independently;
SEQ ID NO: 49 and SEQ ID NO: the concentration of the primers shown by 50 is 0.19-0.252 mu M independently;
SEQ ID NO: 51 and SEQ ID NO: 52 is independently 0.42-0.5 mu M;
SEQ ID NO: 53 and SEQ ID NO: 54 is independently 0.267-0.32 mu M;
SEQ ID NO: 55 and SEQ ID NO: 56 is independently 0.052-0.12 mu M;
SEQ ID NO: 57 and SEQ ID NO: the concentration of the primer shown as 58 is 0.078-0.12 mu M independently;
SEQ ID NO: 59 and SEQ ID NO: the concentration of the primers shown as 60 is 0.08-0.133 mu M independently;
SEQ ID NO: 61 and SEQ ID NO: the concentration of the primers shown in 62 is 0.11-0.2 mu M independently;
SEQ ID NO: 63 and SEQ ID NO: the concentration of the primers shown by 64 is 0.074-0.141 mu M independently;
SEQ ID NO: 65 and SEQ ID NO: 66 is independently 0.277-0.345 MuM;
SEQ ID NO: 67 and SEQ ID NO: 68 is independently 0.18-0.26. mu.M.
7. The kit according to claim 4, wherein the PCR reaction premix solution uses deionized water as a solvent and comprises the following components in concentration: 0.19-0.38U/. mu.l of hot start Taq DNA polymerase, 100-200 mM Tris buffer, 100-200 mM KCL, 3.75-7.5 mM MgCl250 to 100mM (NH)4)2SO40.5-1 mu M dNTP, 1000-1500 mM betaine, 0.19-0.38% Triton by volume fraction, 2-3 mg/ml BSA, 5-15% Tween by volume fraction and 2.5-7.5% glycerol by volume fraction.
8. The kit of claim 5, wherein the allelic ladder and the Y chromosome STR locus correspond in relationship to one another as follows:
in the first group: DYS438 corresponds to 9,10,11,12,13, 14; DYS389I corresponds to 10,11,12,13,14, 15; DYS448 corresponds to 16,17,18,19,20,21,22,23, 24; DYS389II corresponds to 24,25,26,27,28,29,30,31,32,33, 34; DYS19 corresponds to 10,11,12,13,14,15,16,17,18,19, 20; y _ GATA _ H4 corresponds to 8,9,10,11,12, 13; DYS 518: 32,33,34,36,37,38,39, 41;
in the second group: DYS460 corresponds to 8,9,10,11,12,13, 14; DYS458 corresponds to 14,15,16,17,18,18.2,19,20,21, 22; DYS437 corresponds to 13,14,15, 16; DYS439 corresponds to 9,10,11,12,13,14,15,; DYS643 corresponds to 8,9,10,11,12,13,14, 16, 17; DYF387S1 corresponds to 33,34,35,36,37,38,39, 40; DYS385 corresponds to 9,10,11,12,13,14,15,16,17,19,20,21,22, 24;
in the third group: DYS393 corresponds to 8,9,10,11,12,13,14, 15, 16; DYS391 corresponds to 5,6,7,8,9,10,11,13,14,15, 16; DYS390 corresponds to 17,18,19,20,21,22,23,24,25,26, 27; DYS635 corresponds to 17,18,19,20,21,22,23,24,25, 26; DYS449 corresponds to 22,24,25,27,30,31,32,33,34,36,37,38,40,41, 42; DYS596 corresponds to 14,15, 16;
in the fourth group: DYS456 corresponds to 13,14,15,16,17, 18; DYS576 corresponds to 11,12,13,14,15,16, 17; DYS549 corresponds to 7,8,9,10,12,15,16, 17; DYS570 corresponds to 10,11,12,13,15,16,17,18,19,20,21,23,24, 25; DYS481 corresponds to 16,17,18,20,21,22,26,27,28,29,30,31, 32; DYS392 corresponds to 7,9,10,11,12,13,14,15, 16; DYS627 corresponds to 16,17,18,19,20,21,22,23,24, 25;
in the fifth group: y-indel corresponds to 1, 2; DYS388 corresponds to 10,11,12,13,14, 15; DYS447 corresponds to 22,23,24,25,26,27,28,29, 30; DYS557 corresponds to 14,15,16,18,19,20, 21; DYS527 corresponds to 13,14,16,17,18,20,21,22,23,24,25,26, 27; DYS533 corresponds to 7,8,9,10,11,12,14,15, 16; DYS444 corresponds to 10,11,12,13,14,15, 16.
9. Use of the primer set of claim 1 or 2 and the kit of any one of claims 3 to 8 for forensic individual identification, forensic DNA database construction, suspect pedigree screening or forensic genetic relationship identification.
10. The application according to claim 9, characterized in that it comprises the following steps:
placing a sample to be detected into an amplification system containing the primer group of claim 1 or 2, performing PCR amplification to obtain an amplification product, and detecting the amplification product;
when the sample to be detected is an original human body sample, the amplification system of the PCR amplification is calculated by 10 mu L and comprises: 2. mu.l of the primer set mixture, 4. mu.l of the PCR reaction premix and 4. mu.l of deionized water;
when the sample to be detected is human genome DNA, the amplification system of PCR amplification is counted by 10 mu L and comprises: 1. mu.l of human genomic DNA, 2. mu.l of the mixture of the primer sets, 4. mu.l of the PCR reaction premix and 3. mu.l of deionized water;
the amplification procedure of the PCR amplification is as follows: 95 deg.C for 5 min; 94 ℃,10 sec, 59 ℃,40 sec, 72 ℃, 45sec, 30 cycles; 60 deg.C, 30 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011621943.8A CN112852970A (en) | 2020-12-31 | 2020-12-31 | Primer group and kit for simultaneously amplifying 37 human Y-STR loci and application of primer group and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011621943.8A CN112852970A (en) | 2020-12-31 | 2020-12-31 | Primer group and kit for simultaneously amplifying 37 human Y-STR loci and application of primer group and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112852970A true CN112852970A (en) | 2021-05-28 |
Family
ID=75999161
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011621943.8A Pending CN112852970A (en) | 2020-12-31 | 2020-12-31 | Primer group and kit for simultaneously amplifying 37 human Y-STR loci and application of primer group and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112852970A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416791A (en) * | 2021-08-23 | 2021-09-21 | 百特元生物科技（北京）有限公司 | Specific amplification primer group for simultaneously amplifying 25 STR loci of human, fluorescence labeling amplification kit, application and method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106164298A (en) * | 2014-02-18 | 2016-11-23 | 伊鲁米那股份有限公司 | Method and composition for DNA pedigree analysis |
| CN108441565A (en) * | 2018-03-13 | 2018-08-24 | 无锡中德美联生物技术有限公司 | The fluorescence labeling composite amplification kit of 37 str locus seats of human Y-chromosome and its application |
| CN109468384A (en) * | 2018-11-15 | 2019-03-15 | 江苏苏博生物医学科技南京有限公司 | Composite amplification detection kit that is a kind of while detecting 45 Y gene seats |
| CN109750110A (en) * | 2019-03-07 | 2019-05-14 | 基点认知技术(北京)有限公司 | The composite amplification reagent kit and its application of 47 mankind's autosomes and Y chromosome locus |
| CN112852971A (en) * | 2020-12-31 | 2021-05-28 | 百特元生物科技（北京）有限公司 | Primer group and kit for simultaneously amplifying 44 human Y-STR loci and application of primer group and kit |
-
2020
- 2020-12-31 CN CN202011621943.8A patent/CN112852970A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106164298A (en) * | 2014-02-18 | 2016-11-23 | 伊鲁米那股份有限公司 | Method and composition for DNA pedigree analysis |
| CN108441565A (en) * | 2018-03-13 | 2018-08-24 | 无锡中德美联生物技术有限公司 | The fluorescence labeling composite amplification kit of 37 str locus seats of human Y-chromosome and its application |
| CN109468384A (en) * | 2018-11-15 | 2019-03-15 | 江苏苏博生物医学科技南京有限公司 | Composite amplification detection kit that is a kind of while detecting 45 Y gene seats |
| CN109750110A (en) * | 2019-03-07 | 2019-05-14 | 基点认知技术(北京)有限公司 | The composite amplification reagent kit and its application of 47 mankind's autosomes and Y chromosome locus |
| CN112852971A (en) * | 2020-12-31 | 2021-05-28 | 百特元生物科技（北京）有限公司 | Primer group and kit for simultaneously amplifying 44 human Y-STR loci and application of primer group and kit |
Non-Patent Citations (1)
| Title |
|---|
| GUANGYAO FAN ET AL.: "Technical note: developmental validation of a novel 41-plex Y-STR system for the direct amplification of reference samples", 《INT J LEGAL MED》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416791A (en) * | 2021-08-23 | 2021-09-21 | 百特元生物科技（北京）有限公司 | Specific amplification primer group for simultaneously amplifying 25 STR loci of human, fluorescence labeling amplification kit, application and method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109439765B (en) | Composite amplification detection kit for simultaneously detecting 60 autosomal and Y chromosome loci | |
| CN109880911B (en) | Composite amplification kit for 25 human chromosome loci and application thereof | |
| McKiernan et al. | Molecular diagnostic applications in forensic science | |
| CN109750110B (en) | Composite amplification kit for 47 human autosomal and Y chromosome loci and application thereof | |
| US8407010B2 (en) | Methods for rapid forensic analysis of mitochondrial DNA | |
| JP5680304B2 (en) | Rapid forensic DNA analysis | |
| CN110607374A (en) | Fluorescence labeling amplification kit for simultaneously amplifying 27 STR loci of human and application thereof | |
| CN109468384B (en) | Composite amplification detection kit for simultaneously detecting 45Y loci | |
| CN109880912B (en) | Composite amplification kit for 44 human Y chromosome loci and application thereof | |
| CN107385064B (en) | Fluorescence labeling composite amplification kit for simultaneously amplifying human autosomal SNP and STR loci and application thereof | |
| WO2017097135A1 (en) | Composite amplification kit for 33 gene loci of human genome, and application thereof | |
| WO2011023131A1 (en) | Composite amplification kits of 20 short tandem repeats | |
| CN104131072A (en) | Method and system for individual recognition and paternity identification of unknown sample | |
| CN107841567A (en) | Composite amplification system, kit and the application of 28 STRs | |
| CN113416791B (en) | Specific amplification primer group for simultaneously amplifying 25 STR loci of human, fluorescence labeling amplification kit, application and method | |
| CN111088329A (en) | Fluorescence composite amplification system, kit and application thereof | |
| CN102250883A (en) | Fluorescent labeled X-STR gene locus multiplex PCR method and application thereof | |
| CN112852971A (en) | Primer group and kit for simultaneously amplifying 44 human Y-STR loci and application of primer group and kit | |
| CN112852972A (en) | Primer group and kit for simultaneously amplifying 34 STR loci of human and application of primer group and kit | |
| CN110157812A (en) | Composite amplification reagent kit that is a kind of while detecting autosome and Y chromosome str locus seat | |
| CN104031989B (en) | The test kit of the composite amplification of a kind of human gene group DNA 26 locus | |
| CN112852970A (en) | Primer group and kit for simultaneously amplifying 37 human Y-STR loci and application of primer group and kit | |
| CN113584179A (en) | Six-color fluorescence labeling detection system for degrading 64 loci on human autosome and sex chromosome of test material typing | |
| CN109929936B (en) | Fluorescence labeling multiplex amplification kit for detecting human Y chromosome rapid mutation STR locus and application | |
| CN108251537B (en) | Fluorescence labeling composite amplification kit for simultaneously amplifying STR loci of human autosome and Y chromosome and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210528 |