Hao Weiming, Fan Lujuan, Chen Qianqian, Chen Xiaoxiang, Zhang Sichao, Lan Ke, Lu Jian, Zhang Chiyu
Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, China; Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
PLoS One. 2015 Apr 27;10(4):e0123468. doi: 10.1371/journal.pone.0123468. eCollection 2015.
The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3'-5' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3'-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 102 copies and a selectivity of 5 × 10-5 mutant among 107 copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach.
开发简单、准确、快速且经济高效的突变检测技术对于多种遗传疾病、药物遗传学和耐药性的早期诊断及预防至关重要。校对PCR（PR-PCR）于1998年被开发用于突变检测，但由于其等位基因鉴别效率低而很少应用。在此，我们开发了一种改良的PR-PCR方法，使用ddNTP封闭引物以及具有和不具有3'-5'校对功能的DNA聚合酶混合物。ddNTP封闭引物表现出最佳的封闭效率，可避免非特异性引物延伸，而少量高保真DNA聚合酶与常规量Taq DNA聚合酶的混合物提供了最佳的鉴别和扩增效果。改良的PR-PCR方法能够检测各种突变类型，包括点突变和插入/缺失（indels），并且当错配位于ddNTP封闭引物3'端最后八个核苷酸内时允许鉴别扩增。改良的PR-PCR灵敏度为1 - 5×10²拷贝，在10⁷拷贝野生型DNA中对突变体的选择性为5×10⁻⁵。在60份临床血液样本中检测TP53基因的P72R种系突变时，其准确率达100%，并且使用衔接子和融合封闭引物在检测结核分枝杆菌中低频利福平耐药突变方面具有很高潜力。这些结果表明，改良的PR-PCR技术作为一种简单、灵敏且有前景的方法，在检测各种突变或多态性方面是有效的。
