Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe.
The major problem of using somatic mutations as markers of malignancy is that the clinical samples are frequently containing a trace amounts of mutant allele in a large excess of wild-type DNA. Most methods developed thus far for the purpose of tickling this difficult problem require multiple procedural steps that are laborious. We report herein the development of a rapid and simple protocol for detecting a trace amounts of mutant K-ras in a single tube, one-step format. In a capillary PCR, a 17mer peptide nucleic acid (PNA) complementary to the wild-type sequence and spanning codons 12 and 13 of the K-ras oncogene was used to clamp-PCR for wild-type, but not mutant alleles. The designated PNA was labeled with a fluorescent dye for use as a sensor probe, which differentiated all 12 possible mutations from the wild-type by a melting temperature (T(m)) shift in a range of 9 to 16 degrees C. An extension temperature of 60 degrees C and an opposite primer 97 nt away from the PNA were required to obtain full suppression of wild-type PCR. After optimization, the reaction detected mutant templates in a ratio of 1:10,000 wild-type alleles. Using this newly devised protocol, we have been able to detect 19 mutants in a group of 24 serum samples obtained from patients with pancreatic cancer. Taken together, our data suggest that this newly devised protocol can serve as an useful tool for cancer screening as well as in the detection of rare mutation in many diseases.
Luo
Ji-Dung
JD
Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan 333, Taiwan.
Chan
Err-Cheng
EC
Shih
Chun-Liang
CL
Chen
Tai-Long
TL
Liang
Ying
Y
Hwang
Tsann-Long
TL
Chiou
Chiuan-Chian
CC
D015374:Biosensing Techniques; D004247:DNA / Q000097:blood; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D011905:Genes, ras; D006801:Humans; D009154:Mutation; D015341:Nucleic Acid Probes / Q000737:chemistry*; D010190:Pancreatic Neoplasms / Q000175:diagnosis*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013698:Templates, Genetic
D017931:DNA Primers; D015341:Nucleic Acid Probes; D020135:Peptide Nucleic Acids; D004247:DNA