Facilitated detection of oncogene mutations from exfoliated tissue material by a PNA-mediated 'enriched PCR' protocol.
An 'enriched polymerase chain reaction (PCR)' protocol has been established for the sensitive detection of oncogene mutations in body fluid samples from cancer patients. This two-step protocol combines an allele-specific PCR clamping step followed by a PCR-RFLP (restriction fragment length polymorphism) confirmatory step. The method thus resembles a nested PCR technique starting directly from genomic DNA material and, in no more than 54 PCR cycles, allows the sensitive detection of one mutant allele in 10(3) normal alleles. This protocol was tested on bronchial cytology samples and sputum taken from lung cancer patients and point mutations could be detected both in codon 12 of K-ras and in three codons (248, 249, and 273) of the p53 gene. Comparing this protocol with a different 'enriched PCR' method based on repetitive PCR-RFLP steps, a high concordance was noted between the two methods. Although the present protocol seems to be less sensitive by approximately one order of magnitude, it is much easier to perform and thus could be applied to the rapid but sensitive detection of allelic subfractions in a population of cells derived from exfoliative material.
Behn
M
M
Zentrum Innere Medizin, Philipps-Universität Marburg, Baldingerstrasse, D-35033 Marburg, Germany.
Thiede
C
C
Neubauer
A
A
Pankow
W
W
Schuermann
M
M
D001992:Bronchoalveolar Lavage Fluid; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D005240:Feasibility Studies; D016158:Genes, p53 / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D020125:Mutation, Missense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013183:Sputum