Unit

UNIT 25B.7 Representational Difference Analysis

  1. Yuan Chang

Published Online: 1 NOV 2002

DOI: 10.1002/0471142727.mb25b07s60

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Chang, Y. 2002. Representational Difference Analysis. Current Protocols in Molecular Biology. 60:B:25B.7:25B.7.1–25B.7.12.

Author Information

  1. Hillman Cancer Center University of Pittsburgh, Pittsburgh, Pennsylvania

Publication History

  1. Published Online: 1 NOV 2002
  2. Published Print: OCT 2002

Abstract

Representational difference analysis (RDA) couples subtractive hybridization to PCR-mediated kinetic enrichment for the detection of differences between two complex genomes. In this unit, protocols start with the restriction digestion of two comparison DNA samples. Specific linkers are ligated to fragments from each pool and amplicons are generated by PCR. Linkers are removed from both samples and a new linker is added only to size-selected tester amplicons. These tester amplicons are mixed with a large excess of driver amplicons lacking linkers. Hybridization results in three species of dsDNA fragments: (1) both strands derived from driver DNA (lacking linkers on either strand), (2) hybrids with one strand from driver (no linker) and one from tester (with linker), and (3) both strands from tester DNA (linkers on both strands). Excess driver removes DNA fragments common to both samples, and only the DNA fragments unique to the tester are amplified with linker-specific primers.Representational difference analysis (RDA) couples subtractive hybridization to PCR-mediated kinetic enrichment for the detect Representational difference analysis (RDA) couples subtractive hybridization to PCR-mediated kinetic enrichment for the detect