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Genomic Amplification with Transcript Sequencing (GAWTS)

  • Tammy Lind
  • Erik C. Thorland
  • Steve S. Sommer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 65)

Abstract

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).

Keywords

Xylene Cyanol Reverse Transcriptase Buffer Formamide Solution Shadow Band Dideoxynucleotide Sequencing 
These 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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Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Tammy Lind
    • 1
  • Erik C. Thorland
    • 1
  • Steve S. Sommer
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyMayo Clinic/FoundationRochester

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