Detection of low-level KRAS mutations using PNA-mediated asymmetric PCR clamping and melting curve analysis with unlabeled probes.
Detection of somatic mutations in clinical cancer specimens is often hampered by excess wild-type DNA. The aim of this study was to develop a simple and economical protocol without using fluorescent probes to detect low-level mutations. In this study, we combined peptide nucleic acid (PNA)-clamping PCR with asymmetric primers and a melting curve analysis using an unlabeled detection probe. PNA-clamping PCR, which suppressed amplification of the wild-type allele, was more sensitive for KRAS codon 12 mutation detection than nonclamping PCR in 5 different mutant cell lines. Three detection probes were tested (a perfectly matched antisense, a mismatched antisense, and a mismatched sense), and the mismatched sense detection probe showed the highest sensitivity (0.1% mutant detection) under clamping conditions. With this probe, we were able to detect not only the perfectly matched KRAS mutation, but also 4 other mismatched mutations of KRAS. We then applied this protocol to 10 human colon cancer tissues with KRAS codon 12 mutations, successfully detecting the mutations in all of them. Our data indicate that the combination of perfectly matched antisense PNA and a mismatched sense detection probe can detect KRAS mutations with a high sensitivity in both cell lines and human tissues. Moreover, this study might prove an easily applicable protocol for the detection of low-level mutations in other cancer genes.
Oh
Ji Eun
JE
Department of Pathology, College of Medicine, Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea.
Lim
Hee Sun
HS
An
Chang Hyeok
CH
Jeong
Eun Goo
EG
Han
Ji Youn
JY
Lee
Sug Hyung
SH
Yoo
Nam Jin
NJ
D001483:Base Sequence; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D015342:DNA Probes / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics; D016612:Paraffin Embedding; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
D015342:DNA Probes; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D020135:Peptide Nucleic Acids; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins