Prasad D.K. Dhulipala1, Rao S. Takkellapati1, Kate Lavrenov1, Scott Hamilton1, and Jessica Pole2
1 GE Healthcare, Piscataway, NJ, USA
2 Hutchison/MRC Research Centre, University of Cambridge, Cambridge, UK
We present a comparative analysis of the two most commonly used methods for amplification of genomic DNA for use with genomic arrays. Ligation-mediated PCR is one of several current strategies being used for the generation of genomic arrays. In this process, a genomic DNA clone is digested with a restriction enzyme and a universal primer adaptor is ligated to serve as a priming site for PCR amplification. A different method employs degenerate oligonucleotide-primed PCR (DOP-PCR) to randomly amplify bacterial artificial chromosome DNA (BAC DNA). This is accomplished by the use of degenerate primers that selectively amplify human DNA but not contaminating E. coli DNA.
In this protocol, we used GenomiPhi DNA Amplification Kit to representatively amplify BAC DNA and then applied the product to the fabrication of slides for array comparative genomic hybridization (CGH). We present microarray data comparing the performance of the GenomiPhi method against DOP-PCR and demonstrate that the two methods provide data of similar quality. We also show that the GenomiPhi method is less expensive and easier to use than DOP-PCR.
BAC target DNA preparation
The NucleoSpin™ 96 Flash method was used to prepare BAC DNA clones spanning the p12 region of human chromosome 8 and Drosophila genomic regions (as negative controls).
The GenomiPhi DNA Amplification Kit contains four vials with sample buffer, reaction buffer, enzyme mix, and lambda control DNA. In addition, a re