A detection kit for 10 str sites based on high-throughput sequencing
A detection kit and the technology of the kit are applied in the field of gene detection, which can solve the problems of inability to accurately quantify, accurately distinguish, and incapable of solving the STR mutation situation.
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Embodiment 1
[0023] The kit of the present invention consists of:
[0024]
[0025]
[0026] The primer mix was composed as follows:
[0027]
[0028] Detection method:
[0029] 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;
[0030] 2) The above PCR product was purified with 0.9×SPRI magnetic beads and eluted in 20 μl. Qubit Quantitative Concentration.
[0031] 3) Use the KAPA Hyper Prep Kit kit to build a library, and quantify 5 ng of the starting template for library building.
[0032] 4) Quantify the library with KAPA Library Quantification Kit.
[0033] 5) Sequencing with MiSeq, 150SE 10M reads.
[0034] 6) Miseq sequencing data statistics, using Miseq 2x150bp pair-end double-end sequencing, the original read...
Embodiment 2
[0041] Taking D3S1358 as an example to illustrate the primer screening of the kit in Example 1, it is well known in the art that in the detection of STR typing, the primer sequence in the amplification step is very important, and suitable primers will greatly reduce the formation of shadow bands (ie, sutter bands). During the research and development of the present invention, a large number of primers were screened to obtain the primer mixture in Example 1. The specific method is to design different primers to amplify the D3S1358 locus of the same DNA sample, refer to Example 1 for the amplification system and amplification conditions; then analyze the amplified product according to the method of Example 1, and repeat the determination 10 times. The result is as follows:
[0042]
Example 1
Comparative example 1
Comparative example 2
Comparative example 3
Frequency of appearance of shadow bands (times)
0
5
8
4
[0043] Comparati...
Embodiment 3
[0048] STR analysis was performed on parents and children from 10 families, and the detection kits and detection methods refer to Example 1; the results showed that the above STR loci were in line with the Mendelian law of inheritance, and the RCP values were all greater than 99.9999%. It should also be noted that the tested samples were saliva, hair and nails, and all obtained consistent analysis results. And each sample has no shadow peak after PCR amplification and software analysis.
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