Abstract
Determination of transcription initiation sites has commonly been performed by primer extension and RNase protection assay using radioactively labeled oligonucleotides. Recently, a protocol based on modified 5' rapid amplification of cDNA ends (RACE) with the use of fluorescently labeled primer was developed. Here, we describe the use of RACE-based technique in conjunction with the GeneScan analysis for the determination of transcription initiation sites of genes of interest. The RACE technique is based on the ligation of an adapter to both ends of the cDNAs. The gene of interest was first amplified by PCR using a gene-specific and a 5' adapter primer. Subsequently, nested PCR was performed using an internal gene-specific primer paired with a fluorescently end-labeled adapter primer. The size of the fluorescently labeled PCR products was directly determined by the ABI PRISM 377 GeneScan Analyzer. This novel approach provides an accurate, sensitive, and convenient method for mapping transcription initiation sites, especially for genes with multiple transcription initiation sites, for genes expressed at low levels, and for splice variants that display alternative splicing farther than a few hundred nucleotides downstream from the transcription initiation site. This article describes the application of this new method in the mapping of transcription initiation sites of two splice variants of rat frizzled related protein transcripts.