Mutational analysis of p53 in human tumors: direct DNA sequencing and SSCP

This chapter describes the techniques necessary to identify p53 genomic mutations in either frozen or paraffin-embedded tumor samples. DNA is extracted from the tumor samples and then used as a template to amplify the p53 coding sequences by polymerase chain reaction (PCR). The PCR products are characterized by agarose gel electrophoresis. Subsequently, mutations can be detected by direct DNA sequencing of the PCR products, followed by acrylamide gel electrophoresis and autoradiography. Alternatively, PCR products containing mutations can be identified by their aberrant mobility on denaturing acrylamide gels, using single-stranded conformation polymorphism (SSCP).