Message abundance and allele copy number determination using ivt with single-stranded primer-promoter-selector constructs

Abstract

Disclosed is a single stranded primer-promoter-selector construct comprising (in 3' to 5′ orientation) a primer subsequence annealing to the target, a T7 or other promoter subsequence (the template strand), and a selector subsequence. The primer can be extended by template mediated elongation, including reverse transcription, or ligation to another oligonucleotide. The promoter sequence is oriented to direct the in-vitro transcription (IVT) opposite to that of primer extension, where the selector subsequence serves as a template for IVT. The selector is associated with the target subsequence of interest and it, and the amplified product are unique subsequences, dissimilar to other sequence present in the sample. The construct's is useful for determination of the presence and relative abundance of designated subsequences in the sample, multiplex gene expression analysis, multiplex allele counting, determination of polymorphic/mutation site, and loss of heterozygosity.