Accurate quantitation of allele-specific expression patterns by analysis of DNA melting

Abstract

Epigenetic and genetic mechanisms can result in large differences in expression levels of the two alleles in a diploid organism. Furthermore, these differences may be critical to phenotypic variations among individuals. In this study, we present a novel procedure and algorithm to precisely and accurately quantitate the relative expression of each allele. This method uses the differential melting properties of DNAs differing at even a single base pair. By referring to the melting characteristics of the two pure alleles, the fractional contribution of the two alleles to any unknown mixture can be mathematically resolved. These methods are highly accurate and precise because each single melting reaction yields multiple data points for analysis. Finally, we discuss how this approach can be used more generally to accurately quantitate gene expression relative to known standards.