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A Nucleic Acid Mass Spectrometry Parentage Testing Method Based on Informational SNP Set and Its Primers

A technology of paternity identification and nucleic acid mass spectrometry, which is applied in the fields of 30 information SNP marker combinations, primer sequences and paternity identification, can solve the problems of interpretation of results, affect the interpretation of paternity identification results, and difficult DNA typing, etc., and achieve high accuracy high effect

Active Publication Date: 2021-12-07
PRIMBIO GENES BIOTECH WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the wide application of STR loci in forensic identification, its defects are becoming more and more prominent, such as: the high mutation rate of STR loci brings troubles to the interpretation of paternity test results; PCR amplification is not easy to achieve and prevents degradation DNA typing; the limited number of STRs is not conducive to the identification of complex genetic relationships
In addition, because the sequence lengths of different alleles of STR are similar, this will lead to a high error rate of 1%-5%, which will directly affect the interpretation of paternity test results

Method used

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  • A Nucleic Acid Mass Spectrometry Parentage Testing Method Based on Informational SNP Set and Its Primers
  • A Nucleic Acid Mass Spectrometry Parentage Testing Method Based on Informational SNP Set and Its Primers
  • A Nucleic Acid Mass Spectrometry Parentage Testing Method Based on Informational SNP Set and Its Primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Selection of 30 informative SNP markers

[0017] The selection of the 30 information SNP markers used in the paternity testing system in the present invention is based on the data analysis of 2100 individuals mainly from African, European, American and Asian populations, and has gone through three processes, specifically as follows:

[0018] 1) The number of SNPs with heterozygous rate>0.45 and Fst<0.01 was initially screened out from the population data SNPs is 2723;

[0019] 2) On the basis of selecting high heterozygosity rate and low Fst index, not selecting SNPs with a position distance of less than 1Mb, and not selecting sites on the X or Y chromosomes, 195 SNPs for primary screening were obtained;

[0020] 3) SNPs with heterozygosity rate>0.4, population genetic index Fst<0.06 and mutation frequency<0.01% were screened among 195 SNPs initially screened, and 30 optimal SNPs as shown in Table 1 were selected.

Embodiment 2

[0021] Example 2: Parentage identification based on nucleic acid mass spectrometry detection information SNP set

[0022] 1. Genome Extraction

[0023] In this embodiment, the magnetic bead method is used to extract genomic DNA. The sample is saliva, and 2ml of saliva is collected and stored in a saliva stabilizer. The extraction steps of saliva genomic DNA are as follows:

[0024] 1) Add 450 μl Lysis Buffer V, 450 μl sample (mixture of saliva and saliva preservation solution), 220 μl isopropanol, 20 μl proteinase K and 10 μl nucleic acid sedimentation aid to a 1.5ml centrifuge tube, vortex and mix well, and place the centrifuge tube Water bath at 56°C for 10 minutes, and invert 6-8 times during the period.

[0025] 2) Take out the centrifuge tube, add 20 μl of magnetic beads and vortex slightly for mixing, then place in a water bath at 56°C for 20 minutes, during which time vortex every 2 minutes.

[0026] 3) Take out the centrifuge tube and place it on the magnetic stand f...

Embodiment 3

[0071] Embodiment 3: Application in real groups

[0072] In order to verify the feasibility and accuracy of the entire set of information SNP sets for paternity testing in actual families, 20 groups of samples were collected in this example, of which 10 groups were combinations with a clear father-son relationship, and the remaining 10 groups were unrelated unknown males with any A child's pairing combination. These 20 groups of samples are numbered KY-1~KY-20 respectively. The data obtained in Example 2 were used to infer the parent-child relationship, and the results are shown in Table 10.

[0073] Table 10 Application of 30 informative SNP sets in practical populations

[0074] combination number Cumulative Parentage Index (CPI) Relative Chance of Paternity (RCP) actual parental relationship KY-1 856324110 1 biological KY-2 625341255.97 1 biological KY-3 965423826.91 1 biological KY-4 0 0 Not biological KY-5 0 0 ...

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Abstract

The invention discloses a nucleic acid mass spectrometry paternity identification method based on an information SNP set and its primers. The information SNP set used in the method is based on the data analysis of 40 populations, has a high heterozygosity rate>0.4, and has a population genetic Index Fst<0.06, and low mutation frequency <0.01%, random population matching rate <10 ‑15 , suitable for individual identification and paternity identification, wherein the nucleotide sequences of the upstream primers of the 30 informational SNP markers are shown in SEQ ID NO.1~30, and the nucleotide sequences of the downstream primers are shown in SEQ ID NO.31~ As shown in 60, the nucleotide sequence of the single base extension primer is shown in sequence as SEQ ID NO.61-90. After flight mass spectrometry detection information SNP polymorphism and paternity inference test verification, the identification result is consistent with the actual kinship relationship; the information SNP set provided by the invention is used for human paternity identification, and the result is accurate. The mass spectrometry method is preferably used, which can be fast and high-throughput , Simple genotyping, so as to calculate parentage index and carry out parentage judgment, which has the advantages of high accuracy and low cost.

Description

technical field [0001] The invention relates to a detection system for human paternity identification by using nucleic acid mass spectrometry to detect information SNP markers, in particular to 30 information SNP marker combinations, primer sequences and a parentage identification method. Background technique [0002] Parentage Testing is the identification of the blood relationship between individuals through the detection of human genetic markers and the analysis of genetic laws. Modern paternity testing methods use DNA analysis, currently mainly based on capillary electrophoresis (Capillary Electrophoresis, CE) PCR-STR composite fluorescence amplification detection, which is the application of the second-generation genetic marker short tandem repeat (STR) typing technology , that is, through the detection, typing and statistics of 15-20 STRs on DNA samples to achieve the purpose of calculating the probability of parentage. The STR locus is considered to be the most versa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6888C12Q2600/16C12Q2537/143C12Q2565/627C12Q2531/113
Inventor 容芬
Owner PRIMBIO GENES BIOTECH WUHAN CO LTD
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