MESSAGE ABUNDANCE AND ALLELE COPY NUMBER DETERMINATION USING IVT WITH SINGLE-STRANDED PRIMER-PROMOTER-SELECTOR CONSTRUCTS

摘要

A single stranded primer-promoter-selector construct for detecting thepresence or the relativequantity of a target subsequence in a sample. The construct includes (in 3' to5' orientation) a primersubsequence complimentary to the target, a T7 or other promoter subsequence,and a selectorsubsequence. The primer can be extended by template mediated elongation,including reversetranscription, or by ligation to an oligonucleotide. The promoter is orientedto direct in-vitro transcription(IVT) opposite to that of primer extension, where the selector serves as atemplate for IVT. The selector isassociated with the target of interest and it, and the amplified product, areunique subsequences, dissimilarto other sequences present in the sample. The construct is useful fordetermination of the presence andrelative abundance of the target subsequence in the sample, multiplex geneexpression analysis, multiplexallele counting, determination of polymorphic/mutation site, and loss ofheterozygosity.