Unit

UNIT 15.2 Direct DNA Sequencing of PCR Products

  1. Robert L. Dorit1,
  2. Osamu Ohara2,
  3. Charles B-C. Hwang (double-stranded sequencing)3,
  4. Jae Bum Kim4,
  5. Seth Blackshaw (one-step purification)4

Published Online: 1 NOV 2001

DOI: 10.1002/0471142727.mb1502s56

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Dorit, R. L., Ohara, O., Hwang, C. B.-C., Kim, J. B. and Blackshaw, S. 2001. Direct DNA Sequencing of PCR Products. Current Protocols in Molecular Biology. 56:15.2:15.2.1–15.2.13.

Author Information

  1. 1

    Yale University, New Haven, Connecticut

  2. 2

    Shionogi Research Laboratories, Osaka, Japan

  3. 3

    Harvard Medical School, Boston, Massachusetts

  4. 4

    Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 NOV 2001
  2. Published Print: OCT 2001

Abstract

PCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam-Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by “asymmetric PCR,” where one primer is present in vast excess over the other. This single-stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end-labeled products are also presented.