Genomic Amplification with Transcript Sequencing (GAWTS)

Genomic amplification with transcript sequencing (GAWTS) ( 1 , 2 ) is a generally applicable method for direct sequencing of PCR material. GAWTS is centered around the attachment of a phage promoter sequence (T7, Sp6, or T3) to the 5′-end of one or both PCR primers. The phage promoter sequence allows the PCR product to be transcribed into RNA. Subsequently, the RNA is utilized as a single-stranded template for dideoxynucleotide sequencing with AMV reverse transcriptase (Fig. 1 ).   Fig. 1.  Schematic of GAWTS. GAWTS consists of the following three steps: ( 1 ) PCR, in which one or both oligonucleotides contain a phage promoter in addition to a sequence targeting the primer to the region to be amplified, ( 2 ) transcription with the phage promoter, and ( 3 ) dideoxy sequencing of the transcript with reverse transcriptase that is primed with a nested (internal) oligonucleotide. Reprinted with permission of Academic Press from Sommer et al. ( 2 ).