Detection kit for 38 str alleles based on high-throughput sequencing
A detection kit and kit technology, applied in the field of in-situ gene detection kits, can solve the problems of reducing efficiency, increasing manpower and time costs, and being unable to distinguish
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Embodiment 1
[0024] The kit of the present invention consists of:
[0025] 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
[0026] The primer mix was composed as follows:
[0027]
[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 ...
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 allele typing results of fresh blood, the simulated degradation of vWA, D1S1656, D2S441, D5S2500, D8S1179, D9S925, GATA198B05, DYS391, DYS481 in saliva samples , DYS643 and DYS456 allelic fragments were missing in multiple tests (n>5). The comparison kit is the same as E...
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