Highly parallel and short-acting amplification with locus-specific primers to detect single nucleotide polymorphisms by the DigiTag2 assay.
Highly parallel and short-acting amplification with locus-specific primers to detect single nucleotide polymorphisms by the DigiTag2 assay.
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DOI：
10.1371/journal.pone.0029967
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Tokunaga K
中科院分区：
文献类型：
--
作者：
Nishida N;Mawatari Y;Sageshima M;Tokunaga K
DigiTag 2检测能够使用Kapa 2GFast HotStart DNA聚合酶和新方案分析一组96个SNP，总运行时间约为7小时，比之前的方案缩短了6小时。质量参数（转化率，调用率，再现性和一致性）是在相同的水平时，基因型调用使用以前的协议。采用192对基因座特异性引物的多重PCR可用于DigiTag 2检测中的靶标制备，而无需优化反应条件，并且质量参数与96-plex PCR获得的质量参数具有相同的水平。基因座特异性引物能够在2小时内实现足够的（靶扩增子浓度≥5 nM）和特异性（非预期扩增子浓度<2 nM）扩增，即使以96重或192重形式工作时也能够实现可检测的扩增。改进后的DigiTag 2检测方法将成为一个有效的平台，用于在全基因组关联研究后的复制分析中筛选中等数量的SNP（数十至数百个位点）。此外，基因座特异性引物的高度平行和短效扩增因此可以促进其他基于PCR的测定的广泛应用。
The DigiTag2 assay enables analysis of a set of 96 SNPs using Kapa 2GFast HotStart DNA polymerase with a new protocol that has a total running time of about 7 hours, which is 6 hours shorter than the previous protocol. Quality parameters (conversion rate, call rate, reproducibility and concordance) were at the same levels as when genotype calls were acquired using the previous protocol. Multiplex PCR with 192 pairs of locus-specific primers was available for target preparation in the DigiTag2 assay without the optimization of reaction conditions, and quality parameters had the same levels as those acquired with 96-plex PCR. The locus-specific primers were able to achieve sufficient (concentration of target amplicon ≥5 nM) and specific (concentration of unexpected amplicons <2 nM) amplification within 2 hours, were also able to achieve detectable amplifications even when working in a 96-plex or 192-plex form. The improved DigiTag2 assay will be an efficient platform for screening an intermediate number of SNPs (tens to hundreds of sites) in the replication analysis after genome-wide association study. Moreover, highly parallel and short-acting amplification with locus-specific primers may thus facilitate widespread application to other PCR-based assays.
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