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Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing

A technology of DNA typing and single sperm, which is applied in the field of forensic personal identification and testing, and can solve the problems of unsatisfactory and unavailable DNA content

Inactive Publication Date: 2014-04-30
中国医科大学
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Problems solved by technology

[0004] The purpose of the present invention is to solve the above problems. It mainly solves the technical problem that the DNA content in the prior art cannot meet the requirements of conventional inspection of mixed sperm spots and cannot provide complete genetic information of individuals, and provides a method for accurately separating different individuals in mixed samples. method of sperm re-identification

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  • Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing
  • Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing
  • Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing

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Embodiment

[0052] Single sperm capture mitochondrial DNA typing is a method for distinguishing individuals in mixed sperm spots, which is achieved through the following steps:

[0053] 1. Single sperm capture

[0054] The mixed sperm spot samples were smeared, and a single sperm was collected by using the LMD7000 Leica laser microdissection system (Leica, Germany), and each sperm was collected in a test tube.

[0055] 2. Single sperm DNA extraction

[0056] 1. Add 15 μL 5% Chelex-100, 0.2 μL 10 mg / ml proteinase K (final concentration 100 μg / ml), and 0.7 μL 1M DTT (final concentration 40 mM) to each test tube containing single sperm.

[0057] 2. Oscillate for 10s.

[0058] 3. Digest at 56°C for 1h to 2h.

[0059] 4. Oscillate for 10s.

[0060] 5. Incubate in a boiling water bath for 8 minutes.

[0061] 6. Oscillate for 10s.

[0062] 7. Centrifuge at 13000rpm for 2min.

[0063] 8. Put the supernatant in a 4°C refrigerator for later use.

[0064] 3. Nested amplification of mtDNA HV Ⅰ ...

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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 [0001] The invention relates to a method for identifying and testing mixed spots in sexual crimes, specifically a method for single sperm capturing mitochondrial DNA typing to distinguish individuals in mixed sperm spots, and belongs to the technical field of forensic personal identification and testing applications. Background technique [0002] In sexual crime cases, the victim may be sexually assaulted by two or more men at the same time, so the identification of mixed spots is particularly important. Mixed spots refer to the samples tested from two or more individuals. The physical evidence samples obtained in such cases are all mixed sperm spots. At present, the conventional method is to use the differential cleavage method to remove the female component and then perform autosomal STR typing, and calculate the peak height and peak area of ​​the allele peaks in the mixed spectrum and combine the allele frequency of each locus to estimate the individual ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6848C12Q1/6858C12Q2549/119C12Q2531/113
Inventor 王保捷丁梅张璐王云柯庞灏邢佳鑫宣金锋王春红林子青韩松梁克伟李春梅姚军
Owner 中国医科大学
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