Direct cycle sequencing of mutated alleles detected by PCR single-strand conformation polymorphism analysis

Abstract

A rapid protocol for direct sequencing of a mutated allele, detected by combining polymerase chain reaction with single-strand conformation polymorphism (PCR-SSCP) analysis and cycle sequencing using a thermal cycler, is described. End-labeled radioactive primers were used both for PCR-SSCP analysis for the detection of p53 gene mutation and for cycle sequencing using ΔTaq(TM) Version 2.0 DNA polymerase. The point mutations along the various exons of the p53 gene can be rapidly determined by this sequencing method. This protocol requires only a small amount of DNA template (0.1 μg) and produces sequencing images with low backgrounds and very uniform band intensity. It has also been used successfully in the study of other gene mutations including ras and NF-1 (neurofibromatosis 1) genes.