RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers.
RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers.
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DOI：
10.1186/1472-6750-11-80
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发表时间：
2011-08-10
影响因子：
3.5
通讯作者：
Walder JA
中科院分区：
文献类型：
--
作者：
Dobosy JR;Rose SD;Beltz KR;Rupp SM;Powers KM;Behlke MA;Walder JA
聚合酶链反应（PCR）通常用于在研究和诊断环境中检测核酸序列的存在。虽然经常实现高特异性，但生物学要求有时需要将引物放置在次优位置，这导致引物二聚体形成和/或同源序列错误扩增的问题。深海热球菌（Pyrococcus abyssi（P.a.）使用RNase H2使PCR能够使用含有单个核糖核苷酸残基的封闭引物进行，所述封闭引物通过酶（rhPCR）的切割而活化。在引物与靶DNA杂交后，切割发生在RNA碱基的5 '端。引物首先与靶序列杂交以获得活性的要求消除了引物二聚体的形成，并大大减少了密切相关序列的错误扩增。易断裂连接附近的错配降低了RNA酶H2的切割效率，进一步增加了测定的特异性。当应用于单核苷酸多态性（SNPs）的检测时，发现rhPCR比标准等位基因特异性PCR灵敏得多。一般来说，当错配位于RNA：DNA碱基对时，最好的区分发生。rhPCR消除了引物二聚体的形成，并显著提高了PCR在脱靶扩增方面的特异性。该测定的这些优点应该在具有挑战性的qPCR应用中找到实用性，例如基因分型、高水平多重测定和罕见等位基因检测。
The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.88.16.7276
发表时间：
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DOI：
10.1016/j.bbrc.2007.03.075
发表时间：
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影响因子：
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