Detection of p53 gene mutation in cancer tissues by nonradioactive direct sequencing

A simple procedure for direct sequencing of double-stranded PCR products by the dideoxy-termination method has been developed using biotinylated sequencing primers. Sequences of the p53 gene have been obtained from DNA extracted from frozen and formalin-fixed paraffin-embedded cancer tissues. Detection of sequencing ladders was done with chemiluminescent or colorimetric techniques. Both are highly sensitive, but colorimetric detection is less prone to diffusion artifacts, which are common in G-rich regions. Use of this nonradioactive PCR-sequencing protocol allowed rapid and simple determination of p53 gene alterations in human tumors.