CN108950005A - A kind of the Forensic detection system and its application of 30 SNP sites of autosome first ancestor - Google Patents
A kind of the Forensic detection system and its application of 30 SNP sites of autosome first ancestor Download PDFInfo
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Abstract
The invention belongs to forensic genetic test fields, more particularly to the Forensic detection system for disclosing a kind of 30 first ancestor's SNP sites of autosome, the gene loci which is specifically related to: rs12142199, rs10496971, rs67302, rs728404, rs1012586, rs1399272, rs885479, rs4918664, rs8072587, rs1366220, rs1520523, rs2267666, rs3827760, rs1800498, rs4756, rs4749305, rs12425434, rs7752055, rs1475840, rs595961, r S1205357, rs1453858, rs748144, rs3176921, rs16891982, rs723220, rs830599, rs590086, rs741272 and rs2075509.Detection architecture of the present invention can detecte the DNA sample of a variety of tissues and body fluid, can be used not only for ancestors' information of extrapolated sample people from source, and can disclose the genetic structure of different groups, infer its Population Genetics information.
Description
Technical field
The invention belongs to forensic genetic test fields, and in particular to disclose a kind of first ancestor SNP of autosome 30
The Forensic detection system of point.
Background technique
Currently, concerning foreign affairs, relate to probably case as the trend of cumulative year after year is presented in the quickening of economic globalization and internationalization paces,
How to the sample DNA sample of the unknown source people in scene, first ancestor's information inference of sample people from source, i.e. biogeography ancestors are carried out
Information inference is all the technical problem of urgent need to resolve all the time.The ancestral heredity information of different groups can pass through allele
The marked difference of frequency fully demonstrates, and individual first ancestor's configuration information that these first ancestor's informative sites can be used for sample people from source pushes away
It is disconnected.
Theoretically, in different crowd allelic frequency, there are the molecular genetic markers of larger difference, i.e., in group
The gene loci of differentiated, can be as first ancestor's informative site, that is, ancestors' information genetic marker (ancestry information
Markers, AIMs), these molecular genetic markers can not only disclose the genetic structure of group, and can judge live inspection
Biogeography ancestors' information of material people from source, such as the information in the ethnic or intercontinental source of sample source people.Not only facilitate legal medical expert
The identification of individual is learned, but also the property of case, locking suspicion can be defined by associated with given area, specific nationality
The range of people reduces scope of investigation, and case investigation can also be made actively to improve cracking of cases efficiency from passively becoming.
Single nucleotide polymorphism (single nucleotide polymorphisms, SNPs) on autosome is in people
It is widely distributed in genoid group, contain abundant information, segment to be analyzed is short (< 150 bp), is easy to parting, and mutation rate is lower, and
Using not by Effect of gender, it, using the SNP site of these group's differentiateds, is mesh that many SNP, which are found to have crowd's specificity,
The optimal selection that preceding first ancestor's hereditary information is inferred.
((Next Generation Sequencing, NGS) is also referred to as large-scale parallel sequencing to two generation sequencing technologies
Sample DNA based on synthesis order-checking and chip are sequenced, are cut into target fragment and carry out library preparation, use bridge-type by technology
Polymerase chain reaction (Polymerase Chain Reaction, PCR) or Water-In-Oil PCR carry out template preparation, will be to be measured
DNA segment adds nucleotide marker, and light (change) signal is changed into number using computer photosensitive element or semiconductor technology
Signal generates a continuous DNA sequence and read or record base by chip.Between in the past few decades, new SNPs
Excavation rely primarily on the generation sequencing technologies based on PCR and Capillary Electrophoresis, and SNPs parting then depend on Taqman,
The technologies such as Sequenom, SNaPshot, Ligase detection reaction and genetic chip.In recent years, not with two generation sequencing technologies
Disconnected progress, high-throughput and low cost feature is more obvious, thus is widely applied in the parting detection field of SNPs.This
Invention is the platform being sequenced in synthesis based on two generation sequencing technologies, establishes a kind of 30 SNP sites of autosome first ancestor
Forensic detection system, and study its application in medical jurisprudence.
Summary of the invention
The purpose of the present invention is disclose a kind of Forensic detection system of 30 first ancestor's SNP sites of autosome, the system
Can synchronous amplification detect 30 first ancestor's SNP sites, 30 site informations are as follows: rs12142199, rs10496971, rs67302,
rs728404、rs1012586、rs1399272、rs885479、rs4918664、rs8072587、rs1366220、
rs1520523、rs2267666、rs3827760、rs1800498、rs4756、rs4749305、rs12425434、
rs7752055、rs1475840、rs595961、rs1205357、rs1453858、rs748144、rs3176921、
Rs16891982, rs723220, rs830599, rs590086, rs741272 and rs2075509.
In one embodiment of the invention, the PCR amplification system of the system includes two parts, first step amplification packet
Contain: 5 x Phusion HF Buffer, dNTPs mixture, PCR primer mixture, II DNA of Phusion HotStart are poly-
Synthase, template DNA, distilled water.Second step amplification include: 5 x Phusion HF Buffer, dNTPs mixture, Barcode,
PE, II archaeal dna polymerase of Phusion HotStart, first step pcr amplification product and distilled water.
Detection architecture of the present invention can detecte the DNA sample of a variety of tissues and body fluid, for example originating from human blood,
Saliva, muscle and organs and tissues, hair, vaginal secretion or seminal stain equal samples DNA sample.
When detecting DNA sample, kit amplification of the present invention is divided into the amplification of two steps:
First step amplification, reaction system composition are as follows:
| Reaction system | Volume (μ L) |
| 5×Phusion HF Buffer | 10 |
| DNTPs(2.5mM) | 4.8 |
| Each FP(20nM) | 10 |
| Each RP(20nM) | 10 |
| Phusion HotStart Ⅱ DNA Polymerase | 1 |
| Template DNA | 100ng |
| ddH2O | Up to 50μL |
| Total | 50μL |
Second step amplification, reaction system composition are as follows:
| Reaction system | Volume (μ L) |
| 5×Phusion HF Buffer | 10 |
| DNTPs(2.5mM) | 4.8 |
| Barcode(50 μM) | 2 |
| PE 1.0(50 μM) | 2 |
| Phusion HotStart Ⅱ DNA Polymerase | 1 |
| 1 PCR product of Step | 100ng |
| ddH2O | Up to 50μL |
| Total | 50μL |
It is a further object to provide the detection method of the system, the detection method can be used in single sample
The deduction of first ancestor's information can be used for the genetic structure for disclosing different groups.
Specific step is as follows:
1. first step PCR amplification
The preparation of 1.1 systems
By 100ng DNA profiling and 5 x Phusion HF Buffer, 4.8ul dNTPs mixture of 10ul, 20ul PCR draws
Object mixture, II archaeal dna polymerase of 1ul Phusion HotStart mixing, adds to 50ul with distilled water.
1.2 PCR amplification
PCR response procedures are as follows: 98 DEG C carry out a circulation for initial denaturation 2 minutes；98 DEG C are denaturalized 30 seconds, and 60 DEG C are annealed 1 minute, and 72
DEG C extend 1 minute, carry out 20 circulation；72 DEG C extend 1 circulation of progress in 5 minutes eventually, and 4 DEG C save backup.
2. agarose gel electrophoresis
Prepare 2% Ago-Gel.The 3 μ L of PCR product for taking the first step to expand carries out agarose gel electrophoresis.Voltage 140V, fortune
Row 15 minutes.
3. product purification
3.1 are purified using paramagnetic particle method, vortex oscillation AMPure 20 seconds, mix it thoroughly for uniform solution；
3.2 the AMPure of 1 times of sample volume is added into PCR pipe, is stored at room temperature 5 minutes after being vortexed concussion 5 seconds；
3.3 are put in PCR pipe on magnetic frame, until magnetic bead adsorbs (about need 5 minutes) completely；
3.4 holding centrifuge tubes are fixed on magnetic frame, are thoroughly discarded solution, are during which avoided contact with magnetic bead；
3.5 continue that centrifuge tube is kept to be fixed on magnetic frame, and 80% ethyl alcohol of the 180 fresh configurations of μ L is added into centrifuge tube；
3.6 holding centrifuge tubes be fixed on magnetic frame, the magnetic bead wait hang adsorb completely (about 30 seconds) afterwards thoroughly discard second
Alcohol；3.7 repeat step 6-7 twice；
3.8 holding centrifuge tubes, which are fixed on to stand on magnetic frame, to be placed 5 minutes, and ethyl alcohol is made to volatilize completely completely；
3.9 remove centrifuge tube from magnetic frame, be added 30 μ L EB(provide for oneself) or deionized water, vortex oscillation keep magnetic bead complete
After being resuspended in eluent, it is placed at room temperature for 5 minutes；
3.10 are put in centrifuge tube on magnetic frame until magnetic bead adsorbs (about need 5 minutes) completely；
3.11 are transferred to 27 μ L clarification eluent in one 1.5 new ml centrifuge tube.
4. second step PCR amplification
The preparation of 4.1 systems
By 100ng pcr amplification product and 5 x Phusion HF Buffer, 4.8ul dNTPs mixture of 10ul, 2ul
The mixing of II archaeal dna polymerase of Barcode, 2ul PE, 1ul Phusion HotStart, adds to 50ul with distilled water.
4.2 PCR amplification
PCR response procedures are as follows: 98 DEG C carry out a circulation for initial denaturation 2 minutes；98 DEG C are denaturalized 30 seconds, and 65 DEG C are annealed 30 seconds, and 72
DEG C extend 30 seconds, carry out 10 circulation；72 DEG C extend 1 circulation of progress in 5 minutes eventually, and 4 DEG C save backup.
5. agarose gel electrophoresis
Prepare 2% Ago-Gel.Second step amplification 3 μ L of PCR product is taken to carry out gel electrophoresis.Voltage 140V is run 15 minutes.
6. product purification
With step 3 product purification.
7. Concentration Testing
7.1 two Qubit pipes of preparation prepare a Qubit pipe for configuring Standards, each sample；
7.2 configure Quant-iT Working according to Quant-iT Buffer and Quant-iT reagent 200:1
Solution；Each sample and Standard need 200 μ L Working Solution.
Configuration is carried out according to the following table:
| Reaction system | Standards pipe | Sample tube |
| Working Solution is added | 190μL | 198μL |
| Standard is added | 10μL | — |
| Sample to be tested is added | — | 2μL |
| Total volume | 200μL | 200μL |
7.3 vortex oscillations 2-3 seconds, are stored at room temperature 2 minutes.
7.4, which insert a tube into Qubit fluorometer, is read, and reading is actual concentrations multiplied by 10.Library concentration
It is maintained at 10ng/ul or more.
Machine testing on 8.
Using 500 platform of Illumina Nextseq for being sequenced.
The present invention relates to the Forensic detection systems for disclosing a kind of 30 first ancestor's SNP sites of autosome, including amplification body
System, sequencing result assessment and each SNP site genotyping result.The present invention is directed to selected site design primer, and designs compound
Different primers are mixed amplification in same amplification system, once obtain multiple amplified productions by amplification system；And utilized for two generations
Sequencing technologies, compared to traditional Capillary Electrophoresis, the detection flux is higher, and can provide higher accuracy rate and deeper cover
Lid.
Detailed description of the invention
Fig. 1: the site rs595961 sequencing result example in embodiment 1.
Fig. 2: the site rs3827760 sequencing result example in embodiment 1.
Fig. 3: 30 first ancestor's SNP sites of autosome are used to infer first ancestor's information in single sample (embodiment one) sample source.
Specific embodiment
It is illustrated the present invention below in conjunction with attached drawing and further detailed description.It should be pointed out that following
Illustrate to be only to claimed technical solution for example, not to any restrictions of these technical solutions.
Protection scope of the present invention be subject to the appended claims record content.
Embodiment 1
Kit forms of the present invention:
First step amplification, reaction system composition are as follows:
| Reaction system | Volume (μ L) |
| 5×Phusion HF Buffer | 10 |
| DNTPs(2.5mM) | 4.8 |
| Each FP(20nM) | 10 |
| Each RP(20nM) | 10 |
| Phusion HotStart Ⅱ DNA Polymerase | 1 |
| Template DNA | 100ng |
| ddH2O | Up to 50μL |
| Total | 50μL |
Second step amplification, reaction system composition are as follows:
| Reaction system | Volume (μ L) |
| 5×Phusion HF Buffer | 10 |
| DNTPs(2.5mM) | 4.8 |
| Barcode(50 μM) | 2 |
| PE 1.0(50 μM) | 2 |
| Phusion HotStart Ⅱ DNA Polymerase | 1 |
| 1 PCR product of Step | 100ng |
| ddH2O | Up to 50μL |
| Total | 50μL |
Detection method:
1. first step PCR amplification
The preparation of 1.1 systems
By 100ng DNA profiling and 5 x Phusion HF Buffer, 4.8ul dNTPs mixture of 10ul, 20ul PCR draws
Object mixture, II archaeal dna polymerase of 1ul Phusion HotStart mixing, adds to 50ul with distilled water.
1.2 PCR amplification
PCR response procedures are as follows: 98 DEG C carry out a circulation for initial denaturation 2 minutes；98 DEG C are denaturalized 30 seconds, and 60 DEG C are annealed 1 minute, and 72
DEG C extend 1 minute, carry out 20 circulation；72 DEG C extend 1 circulation of progress in 5 minutes eventually, and 4 DEG C save backup.
2. agarose gel electrophoresis
2% Ago-Gel is prepared, first step amplification 3 μ L of PCR product is taken to carry out gel electrophoresis.Voltage 140V runs 15 points
Clock.
3. product purification
3.1 are purified using paramagnetic particle method, vortex oscillation AMPure 20 seconds, mix it thoroughly for uniform solution；
3.2 the AMPure of 1 times of sample volume is added into PCR pipe, is stored at room temperature 5 minutes after being vortexed concussion 5 seconds；
3.3 are put in PCR pipe on magnetic frame, until magnetic bead adsorbs (about need 5 minutes) completely；
3.4 holding centrifuge tubes are fixed on magnetic frame, are thoroughly discarded solution, are during which avoided contact with magnetic bead；
3.5 continue that centrifuge tube is kept to be fixed on magnetic frame, and 80% ethyl alcohol of the 180 fresh configurations of μ L is added into centrifuge tube；
3.6 holding centrifuge tubes be fixed on magnetic frame, the magnetic bead wait hang adsorb completely (about 30 seconds) afterwards thoroughly discard second
Alcohol；
3.7 repeat step 6-7 twice；
3.8 holding centrifuge tubes, which are fixed on to stand on magnetic frame, to be placed 5 minutes, and ethyl alcohol is made to volatilize completely completely；
3.9 remove centrifuge tube from magnetic frame, be added 30 μ L EB(provide for oneself) or deionized water, vortex oscillation keep magnetic bead complete
After being resuspended in eluent, it is placed at room temperature for 5 minutes；
3.10 are put in centrifuge tube on magnetic frame until magnetic bead adsorbs (about need 5 minutes) completely；
3.11 are transferred to 27 μ L clarification eluent in one 1.5 new ml centrifuge tube.
4. second step PCR amplification
The preparation of 4.1 systems
By 100ng pcr amplification product and 5 x Phusion HF Buffer, 4.8ul dNTPs mixture of 10ul, 2ul
The mixing of II archaeal dna polymerase of Barcode, 2ul PE, 1ul Phusion HotStart, adds to 50ul with distilled water.
4.2 PCR amplification
PCR response procedures are as follows: 98 DEG C carry out a circulation for initial denaturation 2 minutes；98 DEG C are denaturalized 30 seconds, and 65 DEG C are annealed 30 seconds, and 72
DEG C extend 30 seconds, carry out 10 circulation；72 DEG C extend 1 circulation of progress in 5 minutes, 4 DEG C of preservations eventually.
5. agarose gel electrophoresis
Prepare 2% Ago-Gel.Second step amplification 3 μ L of PCR product is taken to carry out gel electrophoresis.Voltage 140V runs 15 points
Clock.
6. product purification
With step 3 product purification.
7. Concentration Testing
7.1 two Qubit pipes of preparation prepare a Qubit pipe for configuring Standards, each sample；
7.2 configure Quant-iT Working according to Quant-iT Buffer and Quant-iT reagent 200:1
Solution；Each sample and Standard need 200 μ L Working Solution.
Configuration is carried out according to the following table:
| Reaction system | Standards pipe | Sample tube |
| Working Solution is added | 190μL | 198μL |
| Standard is added | 10μL | — |
| Sample to be tested is added | — | 2μL |
| Total volume | 200μL | 200μL |
7.3 vortex oscillations 2-3 seconds, are stored at room temperature 2 minutes.
7.4, which insert a tube into Qubit fluorometer, is read, and reading is actual concentrations multiplied by 10.Library concentration
It is maintained at 10ng/ul or more.
Machine testing on 8.
Using 500 platform of Illumina Nextseq for being sequenced.
Claims (5)
1. a kind of Forensic detection system of 30 first ancestor's SNP sites of autosome, the system can simultaneously parallel augmentation detection this
30 SNP sites, these sites are respectively: rs12142199, rs10496971, rs67302, rs728404, rs1012586,
rs1399272、rs885479、rs4918664、rs8072587、rs1366220、rs1520523、rs2267666、
Rs3827760, rs1800498, rs4756, rs4749305, rs12425434, rs7752055, rs1475840, rs595961,
rs1205357、rs1453858、rs748144、rs3176921、rs16891982、rs723220、rs830599、rs590086、
Rs741272 and rs2075509.
2. the detection architecture according to claim the 1, which is characterized in that the system includes 30 first ancestor's SNP sites
Primer information.
3. the system according to claim the 1, which is characterized in that the PCR amplification system of 30 first ancestor's SNP sites
Comprising two parts, first step amplification includes: 5 x Phusion HF Buffer, dNTPs mixtures, PCR primer mixture,
II archaeal dna polymerase of Phusion HotStart, template DNA, distilled water；Second step amplification includes: 5 x Phusion HF
Buffer, dNTPs mixture, Barcode, PE, II archaeal dna polymerase of Phusion HotStart, first step PCR amplification produce
Object, distilled water.
4. according in claim the 1-3, detection architecture described in any one, which is characterized in that in the amplification system, the
The concentration that one step expands dNTP mixture is 2.5mM, and the concentration of PCR primer mixture is 20nM, and second step expands dNTP mixing
The concentration of object is 2.5mM, and the concentration of Barcode is 50 μM, and the concentration of PE is 50 μM.
5. the detection method of system described in any one, the detection architecture can detecte respectively in a kind of claim the 1-4
The DNA of kind tissue sample sample, and can be used for the deduction of first ancestor's information of single sample, it can also be used to disclose distinct group
The genetic structure and genetic background of body, the specific steps are as follows:
1) first step PCR amplification
1.1) prepared by amplification system
By 100ng DNA profiling and 5 x Phusion HF Buffer, 4.8ul dNTPs mixture of 10ul, 20ul PCR draws
Object mixture, II archaeal dna polymerase of 1ul Phusion HotStart mixing, adds to 50ul with distilled water；
1.2) pcr amplification reaction condition
PCR response procedures are as follows: 98 DEG C carry out a circulation for initial denaturation 2 minutes；98 DEG C are denaturalized 30 seconds, and 60 DEG C are annealed 1 minute, and 72
DEG C extend 1 minute, carry out 20 circulation；72 DEG C extend 1 circulation of progress in 5 minutes eventually, and 4 DEG C save backup；
2) agarose gel electrophoresis
2% Ago-Gel is prepared, takes 3ul first step amplification PCR product to carry out gel electrophoresis, voltage 140V is run 15 minutes；
3) product purification
3.1) it is purified using paramagnetic particle method, vortex oscillation AMPure 20 seconds, mixes it thoroughly for uniform solution；
3.2) AMPure of 1 times of sample volume is added into PCR pipe, is stored at room temperature 5 minutes after being vortexed concussion 5 seconds；
3.3) PCR pipe is put on magnetic frame, until magnetic bead adsorbs (about need 5 minutes) completely；
3.4) it keeps centrifuge tube to be fixed on magnetic frame, thoroughly discards solution, during which avoid contact with magnetic bead；
3.5) continue that centrifuge tube is kept to be fixed on magnetic frame, 80% ethyl alcohol of the 180 fresh configurations of μ L is added into centrifuge tube；
3.6) keep centrifuge tube be fixed on magnetic frame, the magnetic bead wait hang adsorb completely (about 30s) afterwards thoroughly discard ethyl alcohol；
3.7) step 6-7 is repeated twice；
3.8) it keeps centrifuge tube to be fixed on magnetic frame and stands placement 5 minutes, ethyl alcohol is made to volatilize completely completely；
3.9) centrifuge tube is removed from magnetic frame, be added 30 μ L EB(provide for oneself) or deionized water, vortex oscillation keep magnetic bead complete
After being resuspended in eluent, it is placed at room temperature for 5 minutes；
3.10) centrifuge tube is put on magnetic frame until magnetic bead adsorbs (about need 5 minutes) completely；
3.11) 27 μ L clarification eluent is transferred in a 1.5 new ml centrifuge tubes；
4) second step PCR amplification
4.1) prepared by system
By 100ng pcr amplification product and 5 x Phusion HF Buffer, 4.8ul dNTPs mixture of 10ul, 2ul
The mixing of II archaeal dna polymerase of Barcode, 2ul PE, 1ul Phusion HotStart, adds to 50ul with distilled water；
4.2) PCR amplification
PCR response procedures are as follows: 98 DEG C carry out a circulation for initial denaturation 2 minutes, and 98 DEG C are denaturalized 30 seconds, and 65 DEG C are annealed 30 seconds, and 72
DEG C extend 30 seconds, carry out 10 circulation, 72 DEG C eventually extend 5 minutes carry out 1 circulation, 4 DEG C preservation；
5) agarose gel electrophoresis
2% Ago-Gel is prepared, takes 3 ul second steps amplification PCR product to carry out gel electrophoresis, voltage 140V runs 15 points
Clock；
6) product purification
With step 3) product purification；
7) Concentration Testing
7.1) prepare two Qubit pipes and prepare a Qubit pipe for configuring Standards, each sample；
7.2) Quant-iT Working is configured according to Quant-iT Buffer and Quant-iT reagent 200:1
Solution, each sample and Standard need 200 μ L Working Solution；
Configuration is carried out according to the following table:
7.3) it vortex oscillation 2-3 seconds, is stored at room temperature 2 minutes；
7.4) it inserts a tube into Qubit fluorometer to be read, reading is actual concentrations multiplied by 10；Library concentration is kept
In 10ng/ul or more；
8) machine testing on
The sequencing and typing of 30 first ancestor's SNP sites is used for using 500 platform of Illumina Nextseq.
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| CN110257489A (en) * | 2019-06-17 | 2019-09-20 | 南方医科大学 | A kind of detection technique system of 30 Multiple-allele SNP sites based on the sequencing of two generations |
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| CN107012226A (en) * | 2017-04-20 | 2017-08-04 | 司法部司法鉴定科学技术研究所 | A kind of detection kit and its detection method of the SNP site based on high-flux sequence |
| CN107419017A (en) * | 2017-07-25 | 2017-12-01 | 公安部物证鉴定中心 | The method and system for unknown source individual infer in five continents border group source |
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