29 micro-haplotype sites, screening method, multiplex amplification system and application

A compound amplification system and an amplification system technology, which are applied in compound amplification systems and application fields, can solve the problems of indistinguishable individuals with paternal genetic relationship, inability to be successfully detected and typed, inability to distinguish sibling pairs, and the like, Achieve the effect of reducing the amount of DNA, solving identification problems, and fast detection

Active Publication Date: 2018-09-07
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the proportion of DNA from different individuals in the mixed biological specimens differs too much (less than 5% to 10%), due to the influence of factors such as dominant amplification, shadow peak (stutter), and random effects, the DNA components are less Few individuals can be successfully detected and typed
When Y-STR genetic markers are used, the detect...

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  • 29 micro-haplotype sites, screening method, multiplex amplification system and application
  • 29 micro-haplotype sites, screening method, multiplex amplification system and application
  • 29 micro-haplotype sites, screening method, multiplex amplification system and application

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Embodiment 1

[0058] The screening method of 29 new micro-haplotype sites of the present invention comprises the following steps:

[0059] (1) Population data obtained from the Thousand Genomes database;

[0060] (2) Obtain micro-haplotype genotypes and allele frequencies from the population data according to the screening rules, and calculate the detailed process of the forensic science-related parameters of the candidate sites in different populations;

[0061] (3) Further check the sequences where the screened micro-haplotype sites are located, and exclude sites that affect subsequent sequencing typing.

[0062] In step (1), the candidate micro-haplotype sites are screened from the Thousand Genomes database, such as figure 1 as shown, figure 1 It is part of the population data obtained from the Thousand Genomes database.

[0063] In step (2), the screening rule is that the sequence length is less than 50 bp, there are 4 or more alleles, and the frequency of at least 4 alleles is great...

Embodiment 2

[0074] The composite amplification system of 29 micro-haplotype sites of the present invention, the composite amplification system comprises:

[0075] (1) The first round of PCR amplification system

[0076] The first round of PCR amplification system uses the DNA template as the target to amplify, and the primers in the system are designed according to the selected micro-haplotype sites, and the first round of PCR amplification system also includes bridging sequences ;

[0077] (2) The second round of PCR amplification system

[0078] The second-round PCR amplification system uses the dilution of the first-round amplification product as a template to perform the second-round amplification;

[0079] (3) The third round of PCR amplification system

[0080] The third-round PCR amplification system uses the second-round PCR amplification product as a template for third-round amplification, and the primers used in the third-round PCR amplification system are complementary seque...

Embodiment 3

[0111]The present invention discloses the application of the above composite amplification system in the inspection and typing of mixed biological samples, especially the application in the inspection and typing of mixed biological samples from Chinese population. The application method includes the following steps:

[0112] (1) The DNA templates extracted from the mixed biological samples were subjected to the first round of PCR amplification system, the second round of PCR amplification system, and the third round of PCR amplification in the multi-micro-haplotype site complex amplification system, respectively. Amplification of the system;

[0113] (2) Perform massive parallel sequencing on the final amplification results of the multi-micro-haplotype site multiplex amplification system to obtain the average sequencing depth of each micro-haplotype site in the DNA template.

[0114] In step (2), the fastq sequence files obtained after massively parallel sequencing are analyze...

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Abstract

The invention discloses 29 micro-haplotype sites, a screening method, a multiplex amplification system and an application. The screening method comprises the following steps: acquiring population data, acquiring micro-haplotype genotypes and allele frequencies from the population data, calculating forensic related parameters of candidate sites in different populations and excluding sites affectingsubsequent sequencing typing to obtain the 29 micro-haplotype sites; designing primers and amplification conditions according to the 29 micro-haplotype sites, and establishing the multiplex amplification system based on three-round PCR amplification systems. According to the multiplex amplification system, DNA templates extracted from mixed biological samples are amplified, amplification resultsare subjected to large-scale parallel sequencing, value of the sequencing depth of each micro-haplotype site in the DNA templates is obtained, and finally, the purpose of typing the mixed biological samples is achieved. The multiplex amplification system is suitable for large-scale parallel sequencing technology and has the advantages of high detection flux, high detection speed, large quantity ofobtained data, more accurate detection results and the like.

Description

technical field [0001] The invention belongs to forensic material evidence identification, and in particular relates to 29 micro-haplotype sites, a screening method, a compound amplification system and applications. Background technique [0002] Mixed biological samples refer to samples that are mixed with two or more biological components. Mixed biological specimens are a common type of difficult specimens in actual forensic prosecution. With the continuous advancement of scientific and technological means, more and more criminal cases have added the inspection and use of DNA in mixed biological samples. Mixed biological samples may appear in various types of cases, and most of them are sexual assault and violent injury (such as homicide, dismemberment) cases. Suspicious dander, skin swabs, or bodily fluid evidence collected by investigators at the scene often come from different individuals. Therefore, the DNA testing and determination of mixed biological samples plays ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888
Inventor 陈峰陈鹏李开黄惠结俞延芳俞尤嘉毛征生
Owner NANJING MEDICAL UNIV
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