Rapid characterization of mutations in amplified human hprt cDNA by polyacrylamide gel electrophoresis.

Abstract

Compiling hprt mutation spectra involves the isolation and analysis of numerous 6-thioguanine-resistant clones for identifying characteristic point mutations. Since cDNA amplificates are compulsory intermediates in most mutant classification protocols, we suggest their preliminary characterization by polyacrylamide gel electrophoresis for the rapid distinction of clonal and independent mutants and for streamlining mutant analysis procedures. Based on the human hprt cDNA sequence a strategy was developed for mapping missing exons by analytical digests with a small panel of restriction enzymes. In mutant classification schemes, polyacrylamide gel electrophoresis of AluI-digested cDNA amplificates increased the sensitivity for detecting RT-PCR products of reduced size, e.g., in the case of missing exon 5. Restriction analysis of cDNA amplificates from 109 independent mutant clones showed a significant increase of exon loss after NNK induction as compared to spontaneous or BaP-induced mutants. The determination of exon loss from cDNA amplificates, as carried out for 39 independent mutant PCR products, might direct towards the genomic target sequences carrying the point mutations, that caused the aberrant splicing, thus eliminating the need of laborious multiplex PCR comprising all exons. For single-strand conformation polymorphism (SSCP) analysis of five known point mutations, sub-amplificates comprising exons 7 and 8 of hprt cDNA were obtained. After a combined heat and alkali denaturation of the double-stranded PCR products, the samples were separated in pre-cast polyacrylamide gels under non-denaturing conditions. Five known nucleotide substitutions within the amplified region, including the C508T hot spot mutation, resulted in mobility shifts of single-strand bands relative to the wild type pattern.