Use of a PNA probe to block DNA-mediated PCR product formation in prokaryotic RT-PCR
Use of a PNA probe to block DNA-mediated PCR product formation in prokaryotic RT-PCR
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DOI：
10.2144/000112437
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发表时间：
2007-05-01
期刊：
影响因子：
2.7
通讯作者：
Jacobsen, Carsten S.
中科院分区：
文献类型：
--
作者：
Bender, Mikkel;Holben, William E.;Jacobsen, Carsten S.
本文介绍了一种在逆转录PCR（RT-PCR）扩增特定RNA序列时消除DNA介导的PCR产物形成的新方法。该方法通过将序列特异性PNA探针结合到与感兴趣的序列内的PCR引物附着位点之一直接重叠的基因组序列，利用了肽核酸（PNA）/DNA双链体与DNA/DNA双链体相比更高的解链温度值。阻断探针的杂交排除了引物与DNA的连接，而不影响相同引物与逆转录产生的cDNA序列的连接。使用四步PCR循环，其允许PNA探针在引物退火步骤之前在较高温度下与DNA链杂交。
A novel method eliminating DNA-mediated PCR product formation in reverse transcription PCR (RT-PCR) amplification of specific RNA sequences is described. The method exploits the higher melting temperature values of peptide nucleic acid (PNA)/DNA duplexes compared with DNA/DNA duplexes by binding a sequence-specific PNA probe to a genotomic sequence immediately overlapping one of the PCR-primer attachment sites within the sequence of interest. Hybridization of the blocking probe precludes primer attachment to DNA without affecting attachment of the same primer to the reverse transcription-generated cDNA sequence. A four-step PCR cycle is used that allows the PNA probe to hybridize to the DNA strand at a higher temperature just prior to the primer annealing step.