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Genomic amplification with transcript sequencing.

Publication ,  Journal Article
Stoflet, ES; Koeberl, DD; Sarkar, G; Sommer, SS
Published in: Science
January 29, 1988

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.

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Published In

Science

DOI

ISSN

0036-8075

Publication Date

January 29, 1988

Volume

239

Issue

4839

Start / End Page

491 / 494

Location

United States

Related Subject Headings

  • Transcription, Genetic
  • T-Phages
  • Nucleic Acid Amplification Techniques
  • Mutation
  • Molecular Sequence Data
  • Humans
  • Hemophilia A
  • General Science & Technology
  • Factor IX
  • Exons
 

Citation

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Stoflet, E. S., Koeberl, D. D., Sarkar, G., & Sommer, S. S. (1988). Genomic amplification with transcript sequencing. Science, 239(4839), 491–494. https://doi.org/10.1126/science.3340835
Stoflet, E. S., D. D. Koeberl, G. Sarkar, and S. S. Sommer. “Genomic amplification with transcript sequencing.Science 239, no. 4839 (January 29, 1988): 491–94. https://doi.org/10.1126/science.3340835.
Stoflet ES, Koeberl DD, Sarkar G, Sommer SS. Genomic amplification with transcript sequencing. Science. 1988 Jan 29;239(4839):491–4.
Stoflet, E. S., et al. “Genomic amplification with transcript sequencing.Science, vol. 239, no. 4839, Jan. 1988, pp. 491–94. Pubmed, doi:10.1126/science.3340835.
Stoflet ES, Koeberl DD, Sarkar G, Sommer SS. Genomic amplification with transcript sequencing. Science. 1988 Jan 29;239(4839):491–494.
Journal cover image

Published In

Science

DOI

ISSN

0036-8075

Publication Date

January 29, 1988

Volume

239

Issue

4839

Start / End Page

491 / 494

Location

United States

Related Subject Headings

  • Transcription, Genetic
  • T-Phages
  • Nucleic Acid Amplification Techniques
  • Mutation
  • Molecular Sequence Data
  • Humans
  • Hemophilia A
  • General Science & Technology
  • Factor IX
  • Exons