"Hot spots" mutation analysis of p53 gene in gastrointestinal cancers by amplification of naturally occurring and artificially created restriction sites.
We developed a rapid, simple method to detect "hot spot" point mutations of the p53 gene. A DNA fragment from cancer tissue of a surgical specimen was selectively amplified with specific oligonucleotide primers and then digested with restriction enzymes that recognized artificial or naturally occurring restriction sites. To detect mutations in codons 273 and 245, we created artificial Bst U1 and BgI I sites by introducing a single nucleotide mismatch into the respective mutagenesis primers. We used the naturally occurring restriction sites of Msp I, Hae III, and Hha I to detect mutations in codons 248, 249, and 175, respectively. In 74 cases of gastrointestinal cancer, 5 of 35 colorectal cancers showed mutations; 1 of 15 cases of gastric cancers showed mutation; and 1 of 24 hepatocellular carcinomas showed mutation. This nonradioactive method is an accurate and clinically useful way to detect hot-spot point mutations of the p53 gene in gastrointestinal cancers.
Chen
P H
PH
Department of Molecular Medicine and Clinical Pathology, Taipei Municipal Jen-Ai Hospital, Taiwan, ROC.
Lin
S Y
SY
Wang
C K
CK
Chen
Y J
YJ
Chen
T C
TC
Chang
J G
JG
D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D003062:Codon; D015179:Colorectal Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000737:chemistry; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005770:Gastrointestinal Neoplasms / Q000235:genetics*; D016158:Genes, p53; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics
D003062:Codon; D004273:DNA, Neoplasm; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; C018492:GCCNNNNNGGC-specific type II deoxyribonucleases; C037130:GCGC-specific type II deoxyribonucleases; C024403:GGCC-specific type II deoxyribonucleases