A rapid and nonisotopic method for the screening and sequencing of p53 gene mutations in formalin-fixed, paraffin-embedded tumors.

Abstract

Evaluation of the clinical significance of p53 gene mutations requires molecular analysis of primary tumors from large series of patients with long follow-up periods. In this study, we describe a sensitive, rapid, nonisotopic and inexpensive procedure for the polymerase chain reaction (PCR)-single-stranded conformational polymorphism (SSCP) detection and subsequent sequencing of p53 mutations in formalin-fixed and paraffin-embedded tumor (PET) samples. To optimize this method, we used a panel of 34 mutations previously identified in this gene by isotopic PCR-SSCP analysis of frozen colorectal carcinomas. Identical SSCP band shifts were observed in PET sections from each of these frozen tumors, except for one case in which the tumor cell content was probably too low. All of the 34 mutations were detected by use of an optimized minigel SSCP/silver staining procedure. The total PCR-SSCP screening time with use of this protocol was less than 8 hours. An additional improvement was the use of multiplex SSCP to screen two p53 exons (5/7 and 6/8) simultaneously, thus effectively halving the amount of reagents and time required for mutation analysis. To identify the exact nucleotide alterations, mutant single-stranded DNA was excised from silver-stained SSCP gels, reamplified, and used as template in sequencing reactions. The PCR-SSCP procedure we describe can be performed in routine histopathology laboratories, requiring only a thermal cycler and minigel electrophoresis apparatus. Our results demonstrated the feasibility of conducting large retrospective analyses of p53 gene mutation status in archival tumor material. Studies of this kind should allow elucidation of the clinical role of p53 mutation in various human cancer types.