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PCR Methods of Genotyping

1997; Wiley; Volume: 12; Issue: 1 Linguagem: Inglês

10.1002/0471142905.hg0205s12

1934-8266

Thomas J. Hudson, C D Clark, M. Gschwend, Erica Justice‐Higgins,

Molecular Biology Techniques and Applications

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.