Genomic amplification with transcript sequencing.

Published

Journal Article

A sequencing method called genomic amplification with transcript sequencing (GAWTS) is described that is based on amplification with the polymerase chain reaction (PCR). GAWTS bypasses cloning and increases the rate of sequence acquisition by at least fivefold. The method involves the attachment of a phage promoter onto at least one of the PCR primers. The segments amplified by PCR are transcribed to further increase the signal and to provide an abundance of single-stranded template for reverse transcriptase-mediated dideoxy sequencing. An end-labeled reverse transcriptase primer complementary to the desired sequence generates the additional specificity required to generate unambiguous sequence data. GAWTS can be performed on as little as a nanogram of genomic DNA. The rate of GAWTS can be increased by coamplification and cotranscription of multiple regions as illustrated by two regions of the factor IX gene. Since GAWTS lends itself well to automation, further increases in the rate of sequence acquisition can be expected.

Full Text

Duke Authors

Cited Authors

  • Stoflet, ES; Koeberl, DD; Sarkar, G; Sommer, SS

Published Date

  • January 29, 1988

Published In

Volume / Issue

  • 239 / 4839

Start / End Page

  • 491 - 494

PubMed ID

  • 3340835

Pubmed Central ID

  • 3340835

International Standard Serial Number (ISSN)

  • 0036-8075

Digital Object Identifier (DOI)

  • 10.1126/science.3340835

Language

  • eng

Conference Location

  • United States