High fidelity PCR with an off/on switch mediated by proofreading polymerases combining with phosphorothioate-modified primer.
In the initial report, introducing a single phosphorothioate modification at the very 3' terminus of the oligodeoxynucleotide primer has been shown to effectively protect the oligodeoxynucleotide degradation due to the 3' exonuclease activity. In this study, we reported a novel finding that phosphorothioate modification at the 3' end of primers could not only effectively prevent the primer from degradation, but could also mediate an off-switch extension by Pfu polymerase when primers also carry single or multiple mismatched bases located in the first eight bases of the 3' terminus. This suggests that the combination of 3' phosphorothioate-modified primers with exo+ polymerases such as Pfu constituted an on/off switch, which allows perfectly matched primers to be extended but not mismatched primers. Furthermore, we found that polymerases with different fidelities showed different efficiencies in turning off mismatched-primer mediated extension. So we described here a SYBR green-based real-time quantitative PCR assay for the detection of abundance level of gene expression that did not require fluorescently labeled gene-specific probes or complicated primer combinations. The emergence of real-time quantitative RT-PCR technology is thus suited for a diverse application with a need for high-throughput methods to detect and quantify different gene expressions by way of simplicity, versatility, and accuracy, and thus could complement global microarray-based expression profiling strategies.
Yang
Hui-Ling
HL
SNP Institute, Institute of Pharmacy and Pharmacology, Nanhua University, Hengyang 421001, Hunan, China.
Jiang
Hu-Jun
HJ
Fang
Wei-Yi
WY
Xu
Yang-Yan
YY
Li
Kai
K
Zhang
Jia
J
Liao
Duan-Fang
DF
He
Fu-Chu
FC
D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D010710:Phosphates / Q000737:chemistry*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D010710:Phosphates; D004259:DNA-Directed DNA Polymerase; C035638:thiophosphoric acid