CN103757095A - Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing - Google Patents
Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing Download PDFInfo
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- CN103757095A CN103757095A CN201310578684.9A CN201310578684A CN103757095A CN 103757095 A CN103757095 A CN 103757095A CN 201310578684 A CN201310578684 A CN 201310578684A CN 103757095 A CN103757095 A CN 103757095A
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- 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
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- C12Q1/6858—Allele-specific amplification
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Abstract
A method for distinguishing individuals in mixed seminal stain by single sperm capture and mitochondrial DNA typing mainly solves the technical problems in the prior art that DNA content can not meet the requirements of routine mixed seminal stain test and that complete individual genetic information can not be provided. The method is realized by the steps of single sperm capture, DNA extraction from the single sperms, nested amplification on mt DNA HV I zone, DNA sequence analysis of the products from two amplifications and sperm concentration and autosome STR detection. According to the invention, single sperm mitochondrial DNA with personal characteristics is employed as a detection index, the mixed semen from different individuals is distinguished according to individual semen, and then nuclear DNA detection is carried out, thereby successfully solving the problem of recognizing individuals in mixed sample with components from different individuals. The characteristic of abundant mitochondrial DNA content of single sperm is utilized to meet the requirements of mitochondrial DNA detection by PCR technology; and a plurality of sperms with mitochondrial DNA of the same type are collected for realizing nuclear DNA detection, in order to achieve the purpose of individual identification.
Description
Technical field
The present invention relates in a kind of sexual crime, mixed stain be carried out the method for test for identification, is exactly that a kind of monosperm is caught the method that the difference of Mitochondrial DNA somatotype mixes individuality in seminal stain, belongs to medical jurisprudence individual test for identification applied technical field.
Background technology
In sexual crime case, victim may be subject to two or more male sex's sex assault simultaneously, therefore especially aobvious important to the test for identification of mixed stain.Mixed stain refers to tested sample from two or more individualities.The material evidence sample obtaining in this type of case is mixing seminal stain.The method that routine is taked is at present to carry out euchromosome STR somatotype after application of difference cracking process is removed women's composition, by to mixing the medium position peak height at gene peak and the calculating of peak area of spectral pattern and inferring individual genome type in conjunction with the allelic frequency of each locus, can not determine the whole allelotrope that belong to each individuality; The analytical results of Y chromosome STR can improve the individual recognition ability to seminal fluid mixing sample, but because it has the metrapectic feature of fathership, all male sex that same man is have identical Y chromosome STR type, its somatotype result can be used for getting rid of, and can not realize the object of the same each individuality; Unicellular capture technique can obtain unicellular sample.But the core DNA of single spermatid belongs to monoploid, and its DNA content can not meet the requirement of routine inspection, can not provide individual complete genetic information.
In sum, all detection methods that are applied to seminal fluid biased samples more than two people all can not be accomplished definite each individual object at present.
Summary of the invention
The present invention is to address the above problem as object, the DNA content that mainly solves prior art existence can not meet the requirement that routine inspection mixes seminal stain, the technical problem of the complete genetic information of individuality can not be provided, and provide the sperm of Different Individual in a kind of accurate separation biased sample to determine again the individual method of sperm.
For achieving the above object, the present invention adopts following technical proposals: monosperm is caught the difference of Mitochondrial DNA somatotype and mixed method individual in seminal stain, and the method realizes by lower step:
One, monosperm is caught
To mix seminal stain sample smear, application LMD7000 type come card laser microdissection system (Leica, Germany) is got respectively single sperm, and each sperm is collected respectively in test tube.
Two, monosperm DNA extraction
1, in each test tube that contains monosperm, add 15 μ L 5% Chelex-100,0.2 μ L10mg/ml Proteinase K (final concentration 100 μ g/ml), 0.7 μ L 1M DTT(final concentration 40mM).
2, vibration 10s.
3,56 ℃ of digestion 1h～2h.
4, vibration 10s.
5, in boiling water bath, hatch 8min.
6, vibration 10s.
7, the centrifugal 2min of 13000rpm.
8, supernatant is put in 4 ℃ of refrigerators standby.
Three, the nido in mtDNA HVⅠ district amplification
1, amplification for the first time
Primer sequence is:
F?5′-TAC?ACA?ATC?AAA?GAC?GCC?CTC-3′；
R?5′-GGA?TGA?GGC?AGG?AAT?CAA?AGA?C-3′。
Amplification system is 5 μ L, contains: 10 × buffer, 0.5 μ L, 2.5mM dNTP Mix 0.5 μ L, the each 0.15 μ L of 10uMol Primer, Template 0.5 μ L, 1u KOD-Plus-Neo 0.3 μ L, ddH
2o 2.9 μ L.
Cycling condition is 94 ℃ of 2min; 94 ℃ of 20s, 55.5 ℃ of 30s, 68 ℃ of 40s, 35cycles; 68 ℃ of 5min.
Reaction is carried out on 9700 amplification instruments, and amplification object fragment is 1305bp.
2, amplification for the second time.
Primer sequence is:
F?5′-ACC?TGA?ATC?GGA?GGA?CAA?C-3′；
R?5′-CAA?AGA?CAG?ATA?CTG?CGA?CAT-3′）。
Amplification system is 20 μ L, contains: 10 × buffer, 2 μ L, 2.5mM dNTP Mix 2 μ L, the each 0.6 μ L of 10uMol Primer, amplified production 0.5 μ L for the first time, 1u KOD-Plus-Neo1.2 μ L, ddH
2o 13.1 μ L.
Cycling condition is 94 ℃ of 2min; 94 ℃ of 20s, 55.5 ℃ of 30s, 68 ℃ of 30s, 30cycles; 68 ℃ of 5min.
Reaction is carried out on 9700 amplification instruments, and amplification object fragment is 954bp.
Four, the DNA sequence analysis of secondary amplified production
Sequencing analysis ordinary method is carried out.
Five, sperm enrichment and euchromosome STR detect
1, multitube sperm DNA extract identical mtDNA HV I region sequence is collected in same test tube.
2, be incorporated in QIAamp MinElute
tMin Column
3, the centrifugal 1min of 8000rpm.
4, add AW1 500 μ L, the centrifugal 1min of 8000rpm.
5, add AW2 500 μ L, the centrifugal 1min of 8000rpm.
6, the centrifugal 3min of 13000rpm.
7, add 7 μ LAE elutriants, standing 5min.
8, the centrifugal 3min of 13000rpm.
9, the PowerPlex of the elutriant application Promega company of centrifugal rear acquisition
15 normal dyeing str locus seats of 16HS test kit amplification.PCR reaction is carried out on 9700 amplification instruments.Pcr amplification system and cycling condition complete by operational manual.
Amplification system is: 10 μ L(Mix 2 μ L, Primer 1 μ L, DNA elutriant 7 μ L).
Cycling condition is: 96 ℃ of 2min; 94 ℃ of 30s, 60 ℃ of 30s, 70 ℃ of 45s, 10cycles; 90 ℃ of 30s, 60 ℃ of 30s, 70 ℃ of 45s, 19cycles; 60 ℃ of 30min.
Amplified production, through AB-3130 type DNA genetic analyzer electrophoretic separation and laser scanning analysis, is collected electrophoresis information with 3130Data Collection v3.0 software, and Genemapper ID v3.2 software analysis amplified fragments size, obtains STR somatotype result.
Beneficial effect of the present invention and feature
The invention has the beneficial effects as follows, take the monosperm Mitochondrial DNA with personal characteristics as detecting index, the seminal fluid mixture that derives from Different Individual is distinguished to sperm according to individuality, the detection of row core DNA again, can successfully solve at present cannot be to an individual identification difficult problem for Different Individual source component in biased sample.Feature is, utilizes the more feature of content of monosperm Mitochondria DNA, can meet the requirement of round pcr detection line mitochondrial DNA, collects multiple sperms of Mitochondrial DNA homotype and can realize the detection to core DNA, to reach individual identifying purpose.
Accompanying drawing explanation
Fig. 1 is that two semen mixture euchromosome PCR detected results and monosperm are caught the sperm euchromosome STR somatotype result somatotype collection of illustrative plates that rear mtDNA sequencing comes to the same thing.X-coordinate representative " fragment length " in figure; Ordinate zou representative " fluorescence signal intensity ".
Be illustrated as the sleep together detection qualification result of real case of two male sex and women.A is two people's seminal fluid biased samples, 15 STR analytical resultss of euchromosome after difference cracking process is processed, and each locus can detect 1～4 allelotrope peak, cannot judge the individuality source at each allelotrope peak; B and C be for catching through monosperm, after DNA extraction, mtdna sequence analyze, 15 STR somatotype results of euchromosome of the spermatid that mtdna sequence is identical.Confirmed that mixed semen is the combination that comes from the str locus type person respectively with B and C, cracking of cases result has confirmed two male sex suspects.
Embodiment
Embodiment
Monosperm is caught the difference of Mitochondrial DNA somatotype and is mixed method individual in seminal stain, and the method realizes by lower step:
One, monosperm is caught
To mix seminal stain sample smear, application LMD7000 type come card laser microdissection system (Leica, Germany) is got respectively single sperm, and each sperm is collected respectively in test tube.
Two, monosperm DNA extraction
1, in each test tube that contains monosperm, add 15 μ L 5%Chelex-100,0.2 μ L10mg/ml Proteinase K (final concentration 100 μ g/ml), 0.7 μ L 1M DTT(final concentration 40mM).
2, vibration 10s.
3,56 ℃ of digestion 1h～2h.
4, vibration 10s.
5, in boiling water bath, hatch 8min.
6, vibration 10s.
7, the centrifugal 2min of 13000rpm.
8, supernatant is put in 4 ℃ of refrigerators standby.
Three, the nido in mtDNA HVⅠ district amplification
1, amplification for the first time
Primer sequence is:
F?5′-TAC?ACA?ATC?AAA?GAC?GCC?CTC-3′；
R?5′-GGA?TGA?GGC?AGG?AAT?CAA?AGA?C-3′。
Amplification system is 5 μ L, contains: 10 × buffer, 0.5 μ L, 2.5mM dNTP Mix 0.5 μ L, the each 0.15 μ L of 10uMol Primer, Template 0.5 μ L, 1u KOD-Plus-Neo 0.3 μ L, ddH
2o 2.9 μ L.
Cycling condition is 94 ℃ of 2min; 94 ℃ of 20s, 55.5 ℃ of 30s, 68 ℃ of 40s, 35cycles; 68 ℃ of 5min.
Reaction is carried out on 9700 amplification instruments, and amplification object fragment is 1305bp.
2, amplification for the second time.
Primer sequence is:
F?5′-ACC?TGA?ATC?GGA?GGA?CAA?C-3′；
R?5′-CAA?AGA?CAG?ATA?CTG?CGA?CAT-3′）。
Amplification system is 20 μ L, contains: 10 × buffer, 2 μ L, 2.5mM dNTP Mix 2 μ L, the each 0.6 μ L of 10uMol Primer, amplified production 0.5 μ L for the first time, 1u KOD-Plus-Neo 1.2 μ L, ddH
2o 13.1 μ L.
Cycling condition is 94 ℃ of 2min; 94 ℃ of 20s, 55.5 ℃ of 30s, 68 ℃ of 30s, 30cycles; 68 ℃ of 5min.
Reaction is carried out on 9700 amplification instruments, and amplification object fragment is 954bp.
Four, the DNA sequence analysis of secondary amplified production
Sequencing analysis ordinary method is carried out.
Five, sperm enrichment and euchromosome STR detect
1, multitube sperm DNA extract identical mtDNA HV I region sequence is collected in same test tube.
2, be incorporated in QIAamp MinElute
tMin Column
3, the centrifugal 1min of 8000rpm.
4, add AW1 500 μ L, the centrifugal 1min of 8000rpm.
5, add AW2 500 μ L, the centrifugal 1min of 8000rpm.
6, the centrifugal 3min of 13000rpm.
7, add 7 μ LAE elutriants, standing 5min.
8, the centrifugal 3min of 13000rpm.
9, the PowerPlex of the elutriant application Promega company of centrifugal rear acquisition
15 normal dyeing str locus seats of 16HS test kit amplification.PCR reaction is carried out on 9700 amplification instruments.Pcr amplification system and cycling condition complete by operational manual.
Amplification system is: 10 μ L(Mix 2 μ L, Primer 1 μ L, DNA elutriant 7 μ L).
Cycling condition is: 96 ℃ of 2min; 94 ℃ of 30s, 60 ℃ of 30s, 70 ℃ of 45s, 10cycles; 90 ℃ of 30s, 60 ℃ of 30s, 70 ℃ of 45s, 19cycles; 60 ℃ of 30min.
Amplified production, through AB-3130 type DNA genetic analyzer electrophoretic separation and laser scanning analysis, is collected electrophoresis information with 3130 Data Collection v3.0 softwares, and Genemapper ID v3.2 software analysis amplified fragments size, obtains STR somatotype result.
The present invention can also be applied to for whole human body karyocytes in other biased samples, as individual tests for identification such as histocyte, white corpuscles.
Claims (1)
1. monosperm is caught the method that the difference of Mitochondrial DNA somatotype mixes individuality in seminal stain, and the method realizes by lower step:
One, monosperm is caught
To mix seminal stain sample smear, application LMD7000 type come card laser microdissection system (Leica, Germany) is got respectively single sperm, and each sperm is collected respectively in test tube;
Two, monosperm DNA extraction
1, in each test tube that contains monosperm, add 15 μ L 5% Chelex-100,0.2 μ L10mg/ml Proteinase K (final concentration 100 μ g/ml), 0.7 μ L 1M DTT(final concentration 40mM);
2, vibration 10s;
3,56 ℃ of digestion 1h～2h;
4, vibration 10s;
5, in boiling water bath, hatch 8min;
6, vibration 10s;
7, the centrifugal 2min of 13000rpm;
8, supernatant is put in 4 ℃ of refrigerators standby;
Three, the nido in mtDNA HVⅠ district amplification
1, amplification for the first time
Primer sequence is:
F?5′-TAC?ACA?ATC?AAA?GAC?GCC?CTC-3′，R?5′-GGA?TGA?GGC?AGG?AAT?CAA?AGA?C-3′；
Amplification system is 5 μ L, contains: 10 × buffer, 0.5 μ L, 2.5mM dNTP Mix 0.5 μ L, the each 0.15 μ L of 10uMol Primer, Template 0.5 μ L, 1u KOD-Plus-Neo 0.3 μ L, ddH
2o 2.9 μ L;
Cycling condition is 94 ℃ of 2min; 94 ℃ of 20s, 55.5 ℃ of 30s, 68 ℃ of 40s, 35cycles; 68 ℃ of 5min, reaction is carried out on 9700 amplification instruments, and amplification object fragment is 1305bp;
2, amplification for the second time
Primer sequence is:
F?5′-ACC?TGA?ATC?GGA?GGA?CAA?C-3′，R?5′-CAA?AGA?CAG?ATA?CTG?CGA?CAT-3′）；
Amplification system is 20 μ L, contains: 10 × buffer, 2 μ L, 2.5mM dNTP Mix 2 μ L, the each 0.6 μ L of 10uMol Primer, amplified production 0.5 μ L for the first time, 1u KOD-Plus-Neo1.2 μ L, ddH
2o 13.1 μ L;
Cycling condition is 94 ℃ of 2min; 94 ℃ of 20s, 55.5 ℃ of 30s, 68 ℃ of 30s, 30cycles; 68 ℃ of 5min, reaction is carried out on 9700 amplification instruments, and amplification object fragment is 954bp;
Four, the DNA sequence analysis of secondary amplified production, sequencing analysis ordinary method is carried out;
Five, sperm enrichment and euchromosome STR detect
1, multitube sperm DNA extract identical mtDNA HV I region sequence is collected in same test tube;
2, be incorporated in QIAamp MinElute
tMin Column;
3, the centrifugal 1min of 8000rpm;
4, add AW1 500 μ L, the centrifugal 1min of 8000rpm;
5, add AW2 500 μ L, the centrifugal 1min of 8000rpm;
6, the centrifugal 3min of 13000rpm;
7, add 7 μ LAE elutriants, standing 5min;
8, the centrifugal 3min of 13000rpm;
9, the PowerPlex of the elutriant application Promega company of centrifugal rear acquisition
15 normal dyeing str locus seats of 16HS test kit amplification, PCR reaction is carried out on 9700 amplification instruments, and pcr amplification system and cycling condition complete by operational manual;
Amplification system is: 10 μ L(Mix 2 μ L, Primer 1 μ L, DNA elutriant 7 μ L);
Cycling condition is: 96 ℃ of 2min; 94 ℃ of 30s, 60 ℃ of 30s, 70 ℃ of 45s, 10cycles; 90 ℃ of 30s, 60 ℃ of 30s, 70 ℃ of 45s, 19cycles; 60 ℃ of 30min;
Amplified production, through AB-3130 type DNA genetic analyzer electrophoretic separation and laser scanning analysis, is collected electrophoresis information with 3130 Data Collection v3.0 softwares, and Genemapper ID v3.2 software analysis amplified fragments size, obtains STR somatotype result.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105713895A (en) * | 2014-12-03 | 2016-06-29 | 武汉瑞博祥生物科技有限公司 | Use of agarose in DNA extraction, DNA extraction kit and DNA extraction method |
| CN106520945A (en) * | 2016-11-07 | 2017-03-22 | 江苏苏博生物医学股份有限公司 | Next generation sequencing platform-based noninvasive target mitochondrion sequencing method |
| CN109971867A (en) * | 2019-04-25 | 2019-07-05 | 中国医科大学 | A method of identifying the mixing seminal stain from two individuals |
| CN111621552A (en) * | 2019-06-13 | 2020-09-04 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
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| CN102807975A (en) * | 2011-05-30 | 2012-12-05 | 熊慧 | Method for rapidly extracting nucleic acid from biological sample |
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Patent Citations (2)
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| CN102807975A (en) * | 2011-05-30 | 2012-12-05 | 熊慧 | Method for rapidly extracting nucleic acid from biological sample |
| CN102807976A (en) * | 2011-05-30 | 2012-12-05 | 上海源奇生物医药科技有限公司 | Rapid nucleic acid extraction kit and applications thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105713895A (en) * | 2014-12-03 | 2016-06-29 | 武汉瑞博祥生物科技有限公司 | Use of agarose in DNA extraction, DNA extraction kit and DNA extraction method |
| CN106520945A (en) * | 2016-11-07 | 2017-03-22 | 江苏苏博生物医学股份有限公司 | Next generation sequencing platform-based noninvasive target mitochondrion sequencing method |
| CN109971867A (en) * | 2019-04-25 | 2019-07-05 | 中国医科大学 | A method of identifying the mixing seminal stain from two individuals |
| CN111621552A (en) * | 2019-06-13 | 2020-09-04 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
| CN111621552B (en) * | 2019-06-13 | 2022-05-06 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
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