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Detection kit for 40 str alleles based on high-throughput sequencing

A detection kit, the technology of the kit, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to solve the STR variation, inability to distinguish, and reduce efficiency.

Active Publication Date: 2018-08-31
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first-generation sequencing is to sequence the PCR products after electrophoresis. The obtained sequence usually only covers 1X or 2X of the target sequence. The electrophoresis band is compared with the standard marker sequence to determine the typing result, but it cannot solve the internal STR. Variations, such as mutations in some repeating units, such as mitochondrial heterogeneity, which cannot be identified more accurately through low coverage
In addition, first-generation sequencing cannot effectively distinguish the results of mixed amplification, which undoubtedly increases the cost of manpower and time, and reduces efficiency

Method used

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  • Detection kit for 40 str alleles based on high-throughput sequencing
  • Detection kit for 40 str alleles based on high-throughput sequencing
  • Detection kit for 40 str alleles based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The kit of the present invention consists of:

[0026] components

Final concentration

wxya 2 o

To 20μl

FastStart DNA Polymerase PCR buffer (Roche)

1×

dNTP (Roche)

0.2mM

primer mix

0.2μM

FastStart DNA Polymerase (Roche)

1U

BSA (AMRESCO)

0.16mg / ml

[0027] The primer mix was composed as follows:

[0028]

[0029]

[0030] Detection method:

[0031] 1) Take 1 ng / μl of human DNA sample and add it to the kit for mixed PCR amplification. The amplification procedure is as follows: denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds; annealing at 60°C for 1 minute; extension at 70°C for 1 minute; ℃ last extension 60min; a total of 28 cycles;

[0032] 2) The above PCR product was purified with 0.9×SPRI magnetic beads and eluted in 20 μl. Qubit Quantitative Concentration.

[0033] 3) Use the KAPA Hyper Prep Kit kit to build a library, and quantify 5 ng of the starting...

Embodiment 2

[0042] The kit prepared in Example 1 was tested in parallel on the above samples 50 times to investigate the stability and effect of the mixed amplification system of the kit in Example 1. The test showed that in 50 tests, the primers of the mixed amplification system all effectively amplified their target STR sites, and alleles were not deleted. Description The invention relates to primer stabilization and efficiency.

Embodiment 3

[0044] Take 10 human saliva samples and place them at 37°C for two weeks, extract genomic DNA according to the chelex-100 method, use the kit and amplification method of Example 1 to amplify, sequence and allelic typing. And take the same human blood sample (direct detection after sampling) to amplify according to the above method, compare the allelic typing results, the results show that the degraded sample can be successfully amplified and allelic For genotyping detection, the results of degraded saliva samples were consistent with allelic genotyping of fresh blood samples.

[0045] In addition, the present invention uses a comparative kit for the above detection, and the results show that compared with the allelic typing results of fresh blood, D1S1656, D2S441, D5S2500, D8S1179, D9S925, GATA198B05, DXS6800, DXS6795, DXS10135 in simulated degraded saliva samples , DYS481, DYS643 and DYS635 allelic fragments were missing in multiple testing (n>5). The comparison kit is the s...

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Abstract

The invention belongs to the field of gene detection, in particular to a detection kit for 40 STR (Short Tandem Repeat) alleles based on high-throughput sequencing. The detection kit can be used for simultaneously amplifying the following STR loci, and specifically, the STR loci are D1S1656, D2S441, D3S3045, D4S2366, D5S2500, D6S1043, D7S820, D8S1179, D9S925, D10S1435, D11S2368, D12S391, D13S325, D14S608, D15S659, D16S539, D17S1290, D18S535, D19S253, D20S470, D21S1270, GATA198B05, DXS101, DXS6809, DXS10079, DXS10135, DXS10162, DXS6795, DXS6800, DXS9907, DYS390, DYS576, DYS643, DYS437, DYS481, DYS439, DYS635, DYS549, PentaD and CSF1PO.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a high-throughput sequencing-based STR allele detection kit. Background technique [0002] The repeat series is an important part of the eukaryotic genome sequence. The sequences currently used as genetic markers belong to the repeat region of the genome. Among the numerous repeat sequences, short tandem repeats (STR) are currently widely used genetic markers, especially in field of forensic science. STR is composed of tandem repeat units composed of 2-6bp nucleic acid sequence, and its length is generally between tens and hundreds of bases. STRs are widely distributed throughout the human genome, with an average of 10,000 bases presenting one STR. The STR sequence is short and easy to amplify. There is no differential amplification between the two alleles, so it is suitable for trace and degraded samples. Due to its high polymorphism in different individuals, it can e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6888C12Q2600/156C12Q2600/16C12Q2535/122C12Q2531/113
Inventor 严江伟杨猛杨雅冉陈婧赵晶
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION