Multiplex system, method for detecting unbalanced mixed samples through compound system and application of method

A system and technology for testing materials, applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., which can solve the problems of inability to separate female components, practical limitations, and inability to identify individuals.

Active Publication Date: 2019-04-05
SICHUAN UNIV
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Problems solved by technology

But in this method, the female component may not be separated due to the presence of male epithelial cells
In addition, the cells of the components in the mixed sample are directly separated by cell separation technology or microdissection technology, but this method has high requirements on the quality of the sample and experimental equipment, which limits its application and promotion.
[0005] (2) Separation is carried out in the stage of DNA amplification, which is mainly achieved by using special genetic markers, such as the use of male-specific Y-STR genetic markers to analyze the Y chromosome information of male components in mixed male-female samples, but due to These markers are paternally inherited and are haplotypes and cannot be used for direct personal identification or mixed male-male and female-female samples
However, DIP-STR is rarely distributed in the genome, and the STR locus core repeat flanking regions routinely used in forensic DNA analysis do not have DIP distribution, so this marker cannot be compared with existing STR databases, and its practicality is limited
[0007] (3) After the DNA typing results of the mixed samples are obtained, the typing of the components is separated and analyzed. This method mainly relies on forensic statistics and probability software to complete, and is easily affected by the ratio of mixed samples and the amplification process. The impact of random effects
If the unbalanced ratio of the mixed sample is too large, the amplification stage has caused the low-ratio components to be covered by the high-ratio components, and the information of the low-ratio components in the typing map appears too little or even does not show the information of the low-ratio components. The statistical methods and software Use will be restricted
[0008] At present, there are no effective solutions and means for the analysis of unbalanced mixed test materials in practical work, and the separation and analysis of a small amount of components in unbalanced mixed test materials is still under study

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  • Multiplex system, method for detecting unbalanced mixed samples through compound system and application of method
  • Multiplex system, method for detecting unbalanced mixed samples through compound system and application of method
  • Multiplex system, method for detecting unbalanced mixed samples through compound system and application of method

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Embodiment 1

[0097] Example 1 locus screening

[0098] 1. DNA extraction: Use the Biotec Whole Blood Genomic DNA Rapid Extraction Kit to extract the peripheral blood genomic DNA from the collected samples. Please refer to the instruction manual for the specific method; Concentration Dilute the obtained DNA solution with TE to a working solution with a concentration of 1 ng / μL and a volume of 100 μL, and store at 4°C.

[0099] 2. Screening of SNP-STR loci

[0100] (1) Screening principle: The premise that SNP-STR genetic markers can be used to analyze unbalanced mixed samples is that the genotype combination of the main and minor components in the sample at the SNP-STR locus is an effective informative genotype. The effective informative genotype is the genotype of the minor component DNA, which has alleles that do not exist in the major component, and the occurrence probability of which is related to the frequency of the SNP allele. according to figure 2 Four effective information geno...

Embodiment 2

[0109] Embodiment 2 primer design and synthesis

[0110] When designing conventional STR primers, only the amplification of the core repeat region is considered, and the length of the amplicon is the STR typing result of the DNA; the design of ARMS primers for SNP-STR considers that two genetic markers, SNP and STR, can be obtained by one amplification Information. Using ARMS (amplification arrest mutation technique) principle (see image 3 ), the design of primers for SNP-STR loci is mainly divided into two parts: (1) First, use the Primer3 primer online design tool (http: / / primer3.ut.ee) to design common primers, requiring the 3' terminal base of the pre-primer Located on the target SNP, the back primer is located downstream of the STR core repeat region, and the length of the target fragment is less than 550bp; (2) Design the SNP allele-specific primer on the basis of the common primer, and the front primer obtained in (1) Basically modify the 3' terminal base to make it ...

Embodiment 3

[0116] The establishment and optimization of embodiment 3 composite system

[0117] After the design of the primers for the 18 SNP-STR loci is completed, according to the length of the target amplified fragment of each site and the different fluorescent labels of each primer, three panels of the multiplex amplification system are established: Panel-A, Panel-B, and Panel- C, see the specific grouping Figure 4 ; Figure 4 The middle square is one set of primers for a locus, the width of the square corresponds to the amplicon length range of the target site, and the vertical bar at the bottom is the molecular weight internal standard GeneScan TM Fragment distribution of 600LIZ (the shortest fragment is 20bp, the longest fragment is 600bp).

[0118]The concentration of each primer in the primer mixture of the three panels of the SNP-STR multiplex amplification system is shown in Table 3. The PCR reaction system includes: 2×QIAGEN Multiplex PCR Master 5 μL, DNA (1ng / μL) 1 μL, an...

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Abstract

The invention discloses a multiplex system, a method for detecting unbalanced mixed samples through the multiplex system and application of the method. The multiplex system comprises primers for amplifying 18 SNP-STR (single nucleotide polymorphism-short tandem repeat) sites, each site comprises a common reverse primer and two specific forward primers for increasing mutation, and the specific sequences of the primers are as shown in the SEQ ID NO.1-54 in the specification. The SNP-STR (single nucleotide polymorphism-short tandem repeat) multiplex amplification system has the advantages that the operation process is simplified, the system can be widely used on the basis of existing forensic evidence conventional inspection equipment, mixed samples of two persons at a forensic scene are detected, the same STR (short tandem repeat) typing samples are distinguished, non-invasive prenatal diagnosis and non-invasive prenatal paternity test are realized, a new technical means is provided, andclues and evidence are provided for solving a case.

Description

technical field [0001] The invention belongs to the technical field of forensic detection, and in particular relates to a compound system and a method and application thereof for detecting unbalanced mixed test materials. Background technique [0002] Among the various samples that need to be analyzed in forensic physical evidence inspection, mixed samples are one of the most important and common samples. The complexity of the mixed sample increases with the number of constituent individuals, and the difficulty of detection also varies with the proportion of the constituent DNA. [0003] The key to analyzing and identifying mixed samples is to effectively separate the information of different components. At present, there are mainly three methods for analyzing mixed inspection materials in actual case work: [0004] (1) In the DNA extraction stage, the components are separated before testing, such as semen-vaginal secretion mixed samples, the differential lysis method is g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156C12Q2600/172
Inventor 梁伟波张林王莉蹇慧吕梅励
Owner SICHUAN UNIV
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