Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity.
Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity.
复制标题
DOI：
10.1093/nar/gku1048
复制
发表时间：
2015-01
影响因子：
14.9
通讯作者：
Stenman J
中科院分区：
文献类型：
--
作者：
Ho TH;Dang KX;Lintula S;Hotakainen K;Feng L;Olkkonen VM;Verschuren EW;Tenkanen T;Haglund C;Kolho KL;Stenman UH;Stenman J
在这里，我们提供了第一个策略，使用竞争性的可延伸的阻断探针（ExBP）的等位基因特异性引发与上级选择性在逆转录阶段。为了分析高度相似的RNA变体，其序列与特定变体匹配的逆转录酶引物选择性地仅引发该变体，而对替代变体的错配引发通过杂交和随后在该替代变体模板上完全匹配的ExBP的延伸以形成cDNA-RNA杂合体而被抑制。当温度降低到可能发生错配引发的水平时，该杂交体将使替代RNA模板对于逆转录的热启动方案中由特异性引物引发的错配引发不可用。基于ExBP的逆转录试验检测到BRAF和KRAS突变超过野生型RNA的至少1000倍，并且检测在4-log动态范围内呈线性。这种新的策略不仅以极高的选择性揭示了罕见突变的存在或不存在，而且还提供了一种方便的工具，用于准确测定不同环境中的RNA变体，例如等位基因特异性表达的定量。
Here we provide the first strategy to use a competitive Extendable Blocking Probe (ExBP) for allele-specific priming with superior selectivity at the stage of reverse transcription. In order to analyze highly similar RNA variants, a reverse-transcriptase primer whose sequence matches a specific variant selectively primes only that variant, whereas mismatch priming to the alternative variant is suppressed by virtue of hybridization and subsequent extension of the perfectly matched ExBP on that alternative variant template to form a cDNA–RNA hybrid. This hybrid will render the alternative RNA template unavailable for mismatch priming initiated by the specific primer in a hot-start protocol of reverse transcription when the temperature decreases to a level where such mismatch priming could occur. The ExBP-based reverse transcription assay detected BRAF and KRAS mutations in at least 1000-fold excess of wild-type RNA and detection was linear over a 4-log dynamic range. This novel strategy not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression.
登录
查看更多内容
影响因子：
9.3
作者：
Milbury CA;Li J;Makrigiorgos GM
通讯作者：
Makrigiorgos GM
影响因子：
48
作者：
Levesque, Marshall J.;Ginart, Paul;Wei, Yichen;Raj, Arjun
通讯作者：
Raj, Arjun
影响因子：
2.9
作者：
Ayyadevara, S;Thaden, JJ;Reis, RJS
通讯作者：
Reis, RJS
影响因子：
2.7
作者：
Bender, Mikkel;Holben, William E.;Jacobsen, Carsten S.
通讯作者：
Jacobsen, Carsten S.
影响因子：
4.4
作者：
Main, Bradley J.;Bickel, Ryan D.;Nuzhdin, Sergey V.
通讯作者：
Nuzhdin, Sergey V.