Round A/Round B Amplification of DNA for Microarrays

Abstract

The goal of this procedure is to randomly amplify a sample of DNA to achieve the best possible sequence representation for hybridization to microarrays. This protocol has been used successfully to amplify genomic representations starting with <10 ng of DNA. The protocol consists of three sets of enzymatic reactions. In Round A, Sequenase is used to extend randomly annealed primers to generate templates for subsequent PCR. During Round B, a specific primer is used to amplify the previously generated templates. Amplified material can be labeled by adding fluorescent moieties to the nucleic acids. Alternatively, Round C in this protocol can be used to incorporate either aminoallyl-dUTP or Cy-dye-coupled nucleotides during additional PCR cycles. This protocol may be unsuitable for amplifying material smaller than 250 bp because such material will not be amplified uniformly.