A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis.
BACKGROUND
We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer.


METHODS
DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells.


RESULTS
The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%.


CONCLUSIONS
PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.
Nishikawa
Takashi
T
Second Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan. tnishika@mdanderson.org
Maemura
Kentaro
K
Hirata
Ichiro
I
Matsuse
Ryouichi
R
Morikawa
Hiroshi
H
Toshina
Ken
K
Murano
Mitsuyuki
M
Hashimoto
Keiichi
K
Nakagawa
Yoshihito
Y
Saitoh
Osamu
O
Uchida
Kazuo
K
Katsu
Kenichi
K
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D005243:Feces / Q000737:chemistry*; D005260:Female; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D017354:Point Mutation / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004247:DNA