A rapid and simple procedure for the routine detection of ras point mutations in formalin-fixed, paraffin-embedded tissues.
The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 microns tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3-4 days.
Slebos
R J
RJ
Department of Pathology, The Netherlands Cancer Institute, Amsterdam.
Boerrigter
L
L
Evers
S G
SG
Wisman
P
P
Mooi
W J
WJ
Rodenhuis
S
S
D001483:Base Sequence; D004273:DNA, Neoplasm / Q000235:genetics; D005069:Evaluation Studies as Topic; D005557:Formaldehyde; D011905:Genes, ras; D006652:Histological Techniques; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D015345:Oligonucleotide Probes; D010232:Paraffin; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D005557:Formaldehyde; D010232:Paraffin