Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT.
Clin Chem. 2018 May;64(5):801-809. doi: 10.1373/clinchem.2017.282285. Epub 2018 Feb 14.
Allele-specific PCR is an important diagnostic tool that identifies single-nucleotide variants by preferential amplification of a particular allele, using primers that are mismatched to all but one allele variant.
We applied a fluorescent stopped-flow polymerase assay to measure extension rates from oligonucleotide hairpins to simulate primer-template pairs. Under PCR-applicable conditions, reaction rates were recorded in nucleotides per second per polymerase (nt/s/poly). The effects of temperature, potassium chloride, mismatch type, and position were studied with primarily a deletion mutant of () DNA polymerase and 135 oligonucleotide sequences.
Rates at 65 °C were between 205 ± 11 and 177 ± 8 nt/s/poly for matched templates and between 4.55 ± 0.21 and 0.008 ± 0.005 nt/s/poly for 3'-mismatched templates. Although extension rates progressively increased with mismatches further away from the 3' end, rates were still reduced by as much as 84% with a C · C mismatch 6 bases from the 3' end. The optimal extension temperature for matched sequences was 70 °C, shifting to 55-60 °C for 3' mismatches. KCl inhibited mismatch extension. The Michaelis constant () was increased and the apparent unimolecular rate constant (k) decreased for 3' mismatches relative to matched templates.
Although primer extension of mismatches depends on mismatch type and position, variation also depends on local sequence, KCl concentration, and the type of polymerase. Introduction of 3' mismatches reduces the optimal temperature for extension, suggesting higher annealing temperatures for better allele discrimination. Quantitative descriptions of expected specificity in allele-specific PCR provide additional design direction and suggest when other methods (e.g., high-resolution melting analysis) may be a better choice.
等位基因特异性 PCR 是一种重要的诊断工具，通过优先扩增特定等位基因来识别单核苷酸变异，使用与所有等位基因变异均不匹配但与一个等位基因变异匹配的引物。
我们应用荧光停止流动聚合酶测定法来模拟引物-模板对来测量寡核苷酸发夹的延伸速率。在 PCR 适用条件下，以每秒每聚合酶核苷酸数 (nt/s/poly) 记录反应速率。主要使用 () DNA 聚合酶的缺失突变体和 135 个寡核苷酸序列研究了温度、氯化钾、错配类型和位置的影响。
在 65°C 时，匹配模板的速率在 205±11 和 177±8 nt/s/poly 之间，而 3'-错配模板的速率在 4.55±0.21 和 0.008±0.005 nt/s/poly 之间。尽管延伸速率随着与 3' 端的错配距离的增加而逐渐增加，但在距离 3' 端 6 个碱基处的 C·C 错配时，速率仍降低了 84%。匹配序列的最佳延伸温度为 70°C，而 3' 错配时则转移至 55-60°C。KCl 抑制错配延伸。与匹配模板相比，3' 错配的米氏常数 () 增加，表观单分子速率常数 (k) 降低。
尽管错配引物的延伸取决于错配类型和位置，但变化也取决于局部序列、KCl 浓度和聚合酶类型。引入 3' 错配会降低延伸的最佳温度，这表明更高的退火温度可提高等位基因区分度。等位基因特异性 PCR 中特异性的定量描述提供了额外的设计方向，并表明何时其他方法（例如，高分辨率熔解分析）可能是更好的选择。
