Method for separating mixed samples by SNP detection technology

A technology of mixed samples and detection technology, which is applied in the field of mixed sample splitting to achieve the effect of high detection sensitivity

Active Publication Date: 2019-01-29
安塞斯(北京)生物技术有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the above-mentioned technical problems, the present invention provides a method for splitting mixed samples by SNP detection technology, especially in order to solve the problem of mixed samples (especially two people mixed), using a high-throughput sequencing platform to build a library and analyze the SNP set, which can accurately split Divide mixed samples (especially 2 people mix)

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  • Method for separating mixed samples by SNP detection technology
  • Method for separating mixed samples by SNP detection technology
  • Method for separating mixed samples by SNP detection technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Mixed sample ratio split

[0065] 1): Assume that the mixed sample includes two samples a and b, and the mixing ratio is X 0 :(100-X 0 ), X 0 0 >=0;

[0066] 2): Take N SNP sites to detect the mixed sample to obtain the SNP set of the mixed sample, obtain a total of 2N genotypes from the two samples a and b, and obtain the mutation frequency Y of each SNP site;

[0067] 3): For the mutation frequency Y of each SNP site, there are 9 mixing ratios of a and b, as follows:

[0068] a.2*Y=X 1 *GT 0 +(100-X 1 )*GT 0

[0069] b.2*Y=X 2 *GT 0 +(100-X 2 )*GT 1

[0070] c.2*Y=X 3 *GT 0 +(100-X 3 )*GT 2

[0071] d.2*Y=X 4 *GT 1 +(100-X 4 )*GT 0

[0072] e.2*Y=X 5 *GT 1 +(100-X 5 )*GT 1

[0073] f.2*Y=X 6 *GT 1 +(100-X 6 )*GT 2

[0074] g.2*Y=X 7 *GT 2 +(100-X 7 )*GT 0

[0075] h.2*Y=X 8 *GT 2 +(100-X 8 )*GT 1

[0076] i.2*Y=X 9 *GT 2 +(100-X 9 )*GT 2

[0077] Among them, GT 0 Represents no mutation as 0, GT 1 Represents ...

Embodiment 2

[0080] Embodiment 2: Public Security Criminal Evidence Appraisal

[0081] 1. DNA extraction: gDNA extraction of mixed samples of criminal cases.

[0082] 1) Take the mixed sperm spot (or the mixed blood spot on the murder weapon) in the case of sexual assault into a centrifuge tube containing cell lysate. Invert the centrifuge tube 5-6 times to mix.

[0083] 2) Incubate at room temperature for 10 minutes to lyse the cells. Centrifuge at 2000xg for 10 minutes at room temperature.

[0084] 3) Remove the supernatant, leaving about 1.4mL of liquid in the centrifuge tube.

[0085] 4) Add RNase, protein precipitation solution, and centrifuge at 2000xg for 10 minutes.

[0086] 5) Add isopropanol to the supernatant, centrifuge at 2000×g for 1 minute at room temperature, and discard the supernatant.

[0087] 6) Wash with ethanol and dry to obtain DNA.

[0088] 2. Library construction, template preparation

[0089] 1) Configure the DNA targeted amplification reaction system

[0...

Embodiment 3

[0102] Example 3: Non-invasive Fetal Paternity Test

[0103] In the existing forensic physical evidence identification, the identification of parent-child relationship requires the "child", "father", and "mother" to provide their own test materials, such as blood, hair, saliva, oral cells and bones, etc. can be used for parent-child test. materials, the "children" of which need to be independent individuals to be accurately sampled. The technology of this solution can isolate the SNP set of the child from the peripheral blood of the pregnant woman, eliminate the factor that the child is an independent individual, and determine the parent-child relationship as soon as possible. Since the concentration of fetal free DNA (cell-free-fetusDNA) in the peripheral blood free DNA (cell-free DNA) of pregnant women over six weeks is 5% to 15%, the peripheral blood of pregnant women can be regarded as the sample material mixed by two people. The method of splitting mixed samples by SNP d...

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Abstract

The invention discloses a method for separating mixed samples by an SNP detection technology. The method uses a high-throughput gene sequencing platform (NGS) to detect multiple single nucleotide polymorphisms (SNPs) by amplicon sequencing, which can solve the problems that a mixed sample of two persons cannot be separated happened in criminal evidence identification, civil judicial identificationand clinic. The method includes the following technical points: 1) in order to ensure that amplification efficiency of amplicons is uniform and stable, the 219 amplicons are obtained to achieve simultaneous amplification in the same tube after strict condition design and screening; 2) in order to ensure successful amplification of a degraded sample (the length of a DNA fragment being 160 bp), theSNPs are selected as biomarkers, wherein the SNPs are located in the middle of the 140 bp amplicons; and 3) in order to distinguish whether the sample is a sample of one person or a mixed sample of two persons, a mixing proportion of the mixed sample and SNPs genotype sets of the mixed sample of two persons are further separated.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the splitting of mixed samples in forensic DNA individual identification. Background technique [0002] At present, in the field of forensic DNA individual identification and paternity testing, STR is mainly used as a biomarker. With the development of a new generation of high-throughput genetic detection technology, the forensic application of SNP as a biomarker has begun to emerge. [0003] At present, there are three difficult test materials in the field of forensic science: 1. Mixed samples: mixed sperm spots in sexual assault cases, especially sperm spots without sperm, mixed blood spots on murder weapons, etc. The DNA signals of multiple samples interfere with each other, making it difficult to Identify suspects; 2. Degraded samples: old bone samples, highly decayed samples, blister samples, etc., when DNA is degraded to fragments below 200bp, the experimental library construct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6888
CPCC12Q1/6827C12Q1/6869C12Q2535/122
Inventor 曹彦东赵雪莹
Owner 安塞斯(北京)生物技术有限公司
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