Melting temperature analysis as quantitative method for detection of point mutations.
Different methods have been devised to detect point mutations. Some are very sensitive, detecting mutations even in a background of normal tissue, but none provide information about the percentage of cells with mutant DNA. Here we describe an easy, fast and reliable method, melting temperature analysis, which not only detects point mutations but also provides quantitative information on the percentage of cells with mutant DNA. By this method we detected a G-A transition in codon 12 of the K-ras gene in DNA of subjects with colorectal cancer. The K-ras mutation was found in 9/10 bowel cancers and 8/10 normal adjacent samples. It was also detected in 4/7 stool samples from the same patients. In colorectal cancers, the proportion of K-ras mutant cells was variable: in two the mutant/wild-type DNA ratio was 30/70, in three 50/50, and in four 70/30. Melting temperature analysis was sensitive for the detection of point mutations in bowel cancer and also in apparently normal tissue, providing quantitative information about the percentage of cells with mutant DNA.
Porcelli
B
B
Institute of Biochemistry and Enzymology, Siena University, Italy. porcelli@unisi.it
Frosi
B
B
Terzuoli
L
L
Arezzini
L
L
Pagani
R
R
Civitelli
S
S
Tanzini
G
G
Orlando
C
C
Pazzagli
M
M
Marziliano
N
N
Da Prato
L
L
D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods* / Q000706:statistics & numerical data; D004273:DNA, Neoplasm / Q000737:chemistry* / Q000235:genetics*; D005260:Female; D011905:Genes, ras; D006801:Humans; D008297:Male; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013696:Temperature
D004273:DNA, Neoplasm