Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer.
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.
Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, China; Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Weiming
W
Hao
Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, China; Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Lujuan
L
Fan
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Qianqian
Q
Chen
Department of Gynecologic Oncology, Jiangsu Cancer Hospital, Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, Jiangsu, China.
Xiaoxiang
X
Chen
Huzhou Center for Disease Control and Prevention, Huzhou, Zhejiang, China.
Sichao
S
Zhang
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Ke
K
Lan
Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, China; School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China.
Jian
J
Lu
Pathogen Diagnostic Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
Chiyu
C
Zhang
D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry*; D054306:Dideoxynucleotides / Q000737:chemistry*; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017384:Sequence Deletion / Q000235:genetics*; D019914:Taq Polymerase / Q000737:chemistry
D017931:DNA Primers; D054306:Dideoxynucleotides; D004247:DNA; D019914:Taq Polymerase