Unit

UNIT 2.5 PCR Methods of Genotyping

  1. Thomas J. Hudson1,
  2. Chris D. Clark2,
  3. Michele Gschwend2,
  4. Erica Justice-Higgins3

Published Online: 1 MAY 2001

DOI: 10.1002/0471142905.hg0205s12

Current Protocols in Human Genetics

Current Protocols in Human Genetics

How to Cite

Hudson, T. J., Clark, C. D., Gschwend, M. and Justice-Higgins, E. 2001. PCR Methods of Genotyping. Current Protocols in Human Genetics. 12:2.5:2.5.1–2.5.23.

Author Information

  1. 1

    Whitehead Institute, Cambridge, Massachusetts

  2. 2

    Stanford University School of Medicine, Stanford, California

  3. 3

    Massachusetts General Hospital, Charlestown, Massachusetts

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: FEB 1997

Abstract

Two protocols discuss labeling PCR products with radioactive labels. The PCR products are then analyzed on a denaturing polyacrylamide gel and visualized by direct autoradiography. The resulting band pattern is used to define the SSLP genotype of the individual. Another method of genotyping uses a simple silver staining technique to detect PCR-amplified SSLPs electrophoresed in denaturing polyacrylamide gels. In a fourth protocol, multiple unlabeled PCR products from one individual are pooled, separated on a denaturing polyacrylamide gel, transferred to a nylon membrane, and sequentially hybridized to nonradioactive end-labeled probes derived from the primers used to amplify each SSLP marker. The probes are detected on film using chemiluminescence. Support protocols describe the preparation of an M13 sequence ladder size standard and digoxigenin labeling of the probe, both of which are required for the chemiluminescent method.