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 alteration 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 matching normal DNA. This has been done by separating isotopically labeled PCR products on denaturing gels. We recently showed that deletions within BAT-26, a polyadenine tract within the hMSH2 gene, could be used to establish the RER status of tumors without the need for matching normal DNA. We now propose a rapid and nonisotopic method of determining RER status based on PCR-SSCP analysis of the BAT-26 poly(A) tract. Compared 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 RER+ detected in 183 right-sided colonic, 121 gastric, and 123 endometrial carcinomas was 20%, 10%, and 5% respectively, using this method. Frameshift 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- colon and gastric tumors identified by BAT-26 analysis. Our results demonstrate that nonisotopic PCR-SSCP of the poly(A) BAT-26 tract is a specific, sensitive and rapid method for determining the RER status of human tumors.
Iacopetta
B
B
Department of Surgery, University of Western Australia, Nedlands, Australia. bjiac@cyllene.uwa.edu.au
Hamelin
R
R
D004261:DNA Replication; D004273:DNA, Neoplasm / Q000235:genetics*; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018895:Microsatellite Repeats; D051718:MutS Homolog 2 Protein; D009369:Neoplasms / Q000235:genetics*; D010641:Phenotype; D011061:Poly A; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D017384:Sequence Deletion
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D011061:Poly A; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein