Single-Nucleotide Polymorphisms and Other Mismatches Reduce Performance of Quantitative PCR Assays
Single-Nucleotide Polymorphisms and Other Mismatches Reduce Performance of Quantitative PCR Assays
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DOI：
10.1373/clinchem.2013.203653
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发表时间：
2013-10-01
影响因子：
9.3
通讯作者：
Vandesompele, Jo
中科院分区：
文献类型：
--
作者：
Lefever, Steve;Pattyn, Filip;Vandesompele, Jo
背景：基因组测序研究导致已知单核苷酸多态性（SNP）的数量大幅增加。因此，设计退火到缺乏SNP的区域的引物变得具有挑战性。我们研究了不同的定量PCR（qPCR）相关参数，如定量循环（Cq），扩增效率，和reproduciblity.METHODS：我们使用合成模板和引物，以评估在引物退火位点的错配的影响qPCR检测性能。用5种市售的主混合物进行反应。我们研究了引物错配的数量、类型和位置对Cq值、PCR效率、重复性和yield.RESULTS的影响：错配的影响最明显的是错配核苷酸的数量和它们与引物3'端的距离。另外，需要在单个引物中具有>= 4个错配或在一个引物中具有3个错配而在另一个引物中具有2个错配来完全阻断反应。最后，对于单个错配，错配效应的程度是浓度无关的，而随着错配数量的增加，浓度无关性在较高模板浓度下失败。结论：位于3'端>5 bp的单个错配对qPCR扩增具有中等影响，并且可以耐受。这一发现，连同单个错配的浓度独立性和>= 4个错配的PCR反应的完全阻断，可以帮助绘制qPCR反应中的错配行为，并提高具有高SNP密度的序列或序列同源区域的成功引物设计率。(c)2013年美国临床化学协会
BACKGROUND: Genome-sequencing studies have led to an immense increase in the number of known single-nucleotide polymorphisms (SNPs). Designing primers that anneal to regions devoid of SNPs has therefore become challenging. We studied the impact of one or more mismatches in primer-annealing sites on different quantitative PCR (qPCR)-related parameters, such as quantitative cycle (Cq), amplification efficiency, and reproducibility.METHODS: We used synthetic templates and primers to assess the effect of mismatches at primer-annealing sites on qPCR assay performance. Reactions were performed with 5 commercially available master mixes. We studied the effects of the number, type, and position of priming mismatches on Cq value, PCR efficiency, reproducibility, and yield.RESULTS: The impact of mismatches was most pronounced for the number of mismatched nucleotides and for their distance from the 3' end of the primer. In addition, having >= 4 mismatches in a single primer or having 3 mismatches in one primer and 2 in the other was required to block a reaction completely. Finally, the degree of the mismatch effect was concentration independent for single mismatches, whereas concentration independence failed at higher template concentrations as the number of mismatches increased.CONCLUSIONS: Single mismatches located >5 bp from the 3' end have a moderate effect on qPCR amplification and can be tolerated. This finding, together with the concentration independence for single mismatches and the complete blocking of the PCR reaction for >= 4 mismatches, can help to chart mismatch behavior in qPCR reactions and increase the rate of successful primer design for sequences with a high SNP density or for homologous regions of sequence. (c) 2013 American Association for Clinical Chemistry