Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis.
Short oligonucleotide mass analysis (SOMA) is a technique by which small sequences of mutated and wild-type DNA, produced by PCR amplification and restriction digestion, are characterized by HPLC-electrospray ionization tandem mass spectrometry. We have adapted the method to specifically detect two common point mutations at codon 12 of the c-K-ras gene. Mutations in DNA from 121 colon tumor samples were identified by SOMA and validated by comparison with sequencing. SOMA correctly identified 26 samples containing the 12GAT mutation and four samples containing the 12AGT mutation. Sequencing did not reveal mutant DNA in three samples out of the 26 samples shown by SOMA to contain the 12GAT mutation. In these three samples, the presence of mutant DNA was confirmed by SOMA analysis after selective PCR amplification in the presence of BstN1 restriction enzyme. Additional mutations in codons 12 and 13 were revealed by sequencing in 24 additional samples, and their presence did not interfere with the correct identification of G to A or G to T mutations in codon 12. These results provide the basis for a sensitive and specific method to detect c-K-ras codon 12-mutated DNA at levels below 10-12% of wild-type DNA.
Lleonart
Matilde E
ME
Unit of Gene-Environment Interactions, International Agency for Research on Cancer, 69372 Lyon, France.
Ramón y Cajal
Santiago
S
Groopman
John D
JD
Friesen
Marlin D
MD
D000230:Adenocarcinoma / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D011905:Genes, ras; D006801:Humans; D009841:Oligonucleotides / Q000032:analysis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization
D009841:Oligonucleotides