Detection of N-, H-, and KRAS codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and KRAS-specific primer extension.
OBJECTIVE
Mutations of all three RAS genes, N-, H-, and KRAS, are identified mainly in codons 12, 13, and 61 of exons 2 and 3 in human cancers.


METHODS
DNA samples were isolated from 58 oral cancer and 106 colorectal cancer patients. Multiplex amplification of codons 12 and 13 of exon 2 and codon 61 of exon 3 of three RAS genes using two pairs of universal primers for exons 2 and 3 was performed in a single tube. The products were cleaned and split in three tubes. Each was subjected for primer extension using seven different-sized RAS primers for different RAS gene separately to detect base changes in codons 12, 13, and 61 of each RAS gene.


RESULTS
We compared the results with that from direct sequencing for detecting N-, H-, and KRAS mutations in 58 oral cancers and 106 colorectal cancers. The two methods yield identical results, but our method is superior to direct sequencing in terms the amount of work and time required.


CONCLUSIONS
We presented a rapid method to detect codons 12, 13, and 61 mutations of N-, H-, and KRAS genes in human cancers.
Chang
Ya-Sian
YS
Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Yeh
Kun-Tu
KT
Hsu
Nicholas C
NC
Lin
Shu-Hui
SH
Chang
Tien-Jye
TJ
Chang
Jan-Gowth
JG
D000328:Adult; D000368:Aged; D000818:Animals; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics*; D005260:Female; D011905:Genes, ras; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009062:Mouth Neoplasms / Q000235:genetics; D009154:Mutation; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
D003062:Codon; D017931:DNA Primers; D018631:ras Proteins