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High-throughput sequencing-based detection kit for SNP loci and detection method for detection kit

A detection kit and detection method technology, applied in the field of forensic genetics, can solve the problems of limited detection efficiency, unable to meet the needs of forensic individual identification and kinship identification, etc.

Inactive Publication Date: 2017-08-04
ACADEMY OF FORENSIC SCIENCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The screening of the above SNP sites is based on the European population, and the detection efficiency in the Chinese population is limited, and it also cannot meet the needs of forensic individual identification and kinship identification

Method used

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  • High-throughput sequencing-based detection kit for SNP loci and detection method for detection kit
  • High-throughput sequencing-based detection kit for SNP loci and detection method for detection kit
  • High-throughput sequencing-based detection kit for SNP loci and detection method for detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] This example is the typing result of the standard product DNA9948.

[0066] Its detection method is as follows:

[0067] Step 1) Take 1 μL standard product DNA9948 (10ng / μL) for library construction, the PCR system is 20 μL, which contains 5 μL 2.5×PCR reaction buffer II, 2 μL 5× primer mixture, 1 μL Taq polymerase (2U), 1 μL standard Product DNA 9948 and 11 μL deionized water. PCR conditions for library construction: 95°C pre-denaturation for 11 minutes; 94°C for 30s, 60°C for 60s, 70°C for 60s, 22 cycles; 60°C for 30min.

[0068] Step 2) purifying and quantifying the library constructed in step 1). Purification is done using Ampure magnetic beads, and quantification can be done by Qubit or qPCR. This time, qPCR was used to complete the library quantification.

[0069] Step 3) Ligate adapters and sequencing primers to the library quantified in step 2).

[0070] Step 4) Sequence the library prepared in step 3) on a high-throughput sequencing platform (Ion Torrent f...

Embodiment 2

[0081] This example is the application of the SNP site detection kit of the present invention in the identification of suspected mutations in triplet relatives.

[0082] A case of triplet kinship testing involving the biological mother-child (female)-suspect father. The detection method described in Example 1 was adopted for operation.

[0083] STR detection results based on CE technology: 19 autosomal STR loci were detected using the conventional PCR-CE detection kit Goldeye 20A (Beijing Basic Cognitive Biology Co., Ltd.), and it was found that the mother and child were completely identical at the 19 STR loci. According to Mendel's law of inheritance, the suspect father and child had one-step mutations at two STR loci (vWA and FGA). Through calculation, the cumulative parentage index CPI is 378.361. According to the triplet paternity test standard, it is impossible to judge the parent-child relationship between the suspicious father and the child. Supplementary detection Th...

Embodiment 3

[0087] This example is the forensic detection efficiency of the SNP site detection kit of the present invention in 50 unrelated individuals.

[0088] The blood samples of 50 unrelated individuals were tested using the detection method described in Example 1. 50 individuals were volunteers.

[0089] result:

[0090] (1) 50 individuals were completely typed at 234 autosomal SNP loci and 30 X-SNP loci, and 25 male individuals were completely genotyped at 9 Y-SNP loci.

[0091] (2) According to the frequency data of 50 unrelated individuals at 273 SNP sites, it was found that 234 autosomal SNP sites were independent of each other, and the cumulative individual identification rate for individual identification was 1-1.49E-62, which was used for doublet The cumulative non-paternal exclusion rate for body paternity testing is 1-2.98E-12, and the cumulative non-paternal exclusion rate for triplet paternity testing is 1-3.51E-23, which can meet the actual work needs. The detection d...

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Abstract

The invention relates to a high-throughput sequencing-based detection kit for SNP loci used for detecting 273 SNP loci (234 SNP loci are located on an autosome, 9 SNP loci are located on a Y chromosome and 30 SNP loci are located on an X chromosome). The detection kit comprises a primer composition formed by primer pairs of 273 SNP loci. The sequence of the primer composition is as shown in SEQ ID NO:1 to SEQ ID NO:546. The invention further provides a detection method using the kit, and an application of the kit in the field of judicial identification of triplet paternity test, diad paternity test, grandparent and grandchild test, siblings identification and individual recognition. Parallel sequencing is carried out on the SNP loci by adopting a high-throughput sequencing technology, and variation information of a flanking sequence can be obtained while locus information is read; sequence information of 384 samples on the 273 SNP loci can be obtained through single sequencing, so that the DNA sample capacity is reduced and the detection time is shortened; and fragment libraries are concentrated below 200bp and are suitable for forensic degradable materials.

Description

technical field [0001] The invention belongs to the field of forensic genetics and relates to the detection of SNP genetic markers with good forensic application value in the human genome, in particular to a detection kit for 273 SNP sites based on high-throughput parallel sequencing technology and its application and detection method . Background technique [0002] PCR-STR composite fluorescence amplification detection based on capillary electrophoresis (Capillary Electrophoresis, CE) is the main technical means currently used. Among them, the STR locus is considered to be the most versatile genetic marker in forensic DNA identification due to its advantages of high polymorphism and uniform detection technology. Currently, most forensic DNA databases revolve around STR loci. However, with the widespread application of STR loci in forensic DNA analysis, its defects have attracted increasing attention from the academic community. For example, the high mutation rate of STR ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6888C12Q2600/156C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 李成涛张素华边英男刘希玲
Owner ACADEMY OF FORENSIC SCIENCE
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