[Detection of nucleotide mutation by direct sequencing method using non-radio isotopic marker]

We report a protocol which can analyze DNA by the dideoxy method. First, we prepared DNA from paraffin specimen of colon cancer and normal tissue by the method using proteinase and phenol. Polymerase chain reaction (PCR) was performed as follows. The primers used were oligonucleotides corresponding to the sequence of exon 5 on p53. An initial denaturing step was carried out at 94 degrees C for 2 min. Products were amplified for 40 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Specific PCR products derived from p53 gene were purified. Protocol for the PCR-sequencing reaction: The reaction mixture was divided into four 4 microliters fraction. Each fraction was mixed with 2 microliters of NTP solution including non-RI dideoxynucleotides (TOYOBO). PCR was carried out as follows: an initial denaturing step at 94 degrees C for 1 min, then 30 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Prior to loading in a denaturing 8% polyacrylamide-6M Urea gel, the samples were heated to 94 degrees C for 2 min then quickly chilled in ice-water. Electrophoresis was carried out at 1000V for 3hr and transcribed to a nylon membrane. The ladders of DNA were obtained by Non-RI Detection Kit (TOYOBO). We determined the sequence of 167 nucleotides. Results indicated that the point mutations in DNA could be easily detected.