Detection of K-ras oncogene mutations by polymerase chain reaction-based ligase chain reaction.
To evaluate a rapid multiplexed assay to detect three common K-ras codon 12 mutations, primer pairs complementary to the wild-type and mutant loci were developed and tested with lung cancer cell lines with previously identified mutation status. The sensitivity of detection of mutations was determined to be at least 1% using spiked samples containing K-ras codon 12 mutations. This assay was then used to evaluate prospectively K-ras status in airways of individuals at high risk of lung cancer by analysis of bronchoalveolar lavage (BAL) specimens from patients who have been previously treated for lung cancer. DNA was extracted from BAL specimen cell pellets, and PCR-based ligase chain reaction was performed for mutations in the first position of codon 12 of K-ras, with positive and negative controls. Of 10 BAL samples, 4 contained 1 mutation (GGT --> TGT), 1 contained 2 mutations (GGT --> TGT and GGT --> AGT), and the rest were wild-type. The BAL mutations were validated by cloning and screening with mutant-specific probes followed by confirmation sequencing.
Lehman
T A
TA
BioServe Biotechnologies, Ltd., Laurel, Maryland, 20707, USA.
Scott
F
F
Seddon
M
M
Kelly
K
K
Dempsey
E C
EC
Wilson
V L
VL
Mulshine
J L
JL
Modali
R
R
D018893:Bronchoalveolar Lavage; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D003062:Codon / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D011905:Genes, ras / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D014407:Tumor Cells, Cultured
D003062:Codon; D008025:Ligases