A method to detect and quantitate the expression of normal versus mutant h-ras transcripts at codon-12

We describe a PCR based method for qualitative and quantitative analysis of the H-ras gene. For qualitative analysis the aim was to detect codon 12 point mutations at transcriptional and genomic levels by a non-radioactive RFLP assay. Quantitative analysis was achieved by constructing an internal competitor with the same primer site requirements and sequence homology to the H-ras1 gene. The internal control was cloned into an expression vector allowing quantification at the genomic as well as transcriptional level. Two cell lines harbouring normal and T24 H-ras1 respectively, were employed as controls for qualitative and quantitative analysis. Theoretical implications of competitive quantification are also evaluated for increased estimation efficiency.