Abstract

AbstractGenomic 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). 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). KeywordsXylene CyanolReverse Transcriptase BufferFormamide SolutionShadow BandDideoxynucleotide SequencingThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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