RAPID AND NONISOTOPIC SSCP-BASED ANALYSIS OF THE BAT-26 MONONUCLEOTIDE REPEAT FOR IDENTIFICATION OF THE REPLICATION ERROR PHENOTYPE IN HUMAN CANCERS

Microsatellite instability is an important new form of genetic alterat ion that characterizes tumors of the replication error phenotype (RER). The RER status of tumors has been determined until now by analyzing several microsatellite loci for size variations compared with matchin g normal DNA. This has been done by separating isotopically labeled PC R products on denaturing gels. We recently showed that deletions withi n BAT-26, a polyadenine tract within the hMSH2 gene, could be used to establish the RER status of tumors without the need for matching norma l DNA. We now propose a rapid and nonisotopic method of determining RE R status based on PCR-SSCP analysis of the BAT-26 poly(A) tract. Compa red with conventional means of examining RER+, this method reduces the PCR and gel manipulations involved by 10-fold. Size variations of as little as 2 bp in the BAT-26 sequence were readily detected using mini sized, silver-stained SSCP gels run for 2.5 h. The incidence of RERdetected in 183 right-sided colonic, 121 gastric, and 123 endometrial carcinomas was 20%, 10%, and 5% respectively, using this method. Frame shift mutations in the mononucleotide repeat of the TGF-beta RII gene were found in 86% (42/49) of RER+ but less than 1% (1/237) of RER- col on and gastric tumors identified by BAT-26 analysis. Our results demon strate that nonisotopic PCR-SSCP of the poly(A) BAT-26 tract is a spec ific, sensitive and rapid method for determining the RER status of hum an tumors. Hum Mutat 12:355-360, 1998. (C) 1998 Wiley-Liss, Inc.