TRANSCRIPTOME 2002: From Functional Genomics to Systems
Preparation of cDNA Hybridization Targets by Phi29 DNA Polymerase
Rohini Dhulipala, Mubasher Dar, R. Scott Duthie and John Nelson, Amersham Biosciences, Piscataway, NJ
We have investigated a method of cDNA amplification using phi29 DNA polymerase. The unique strand displacement property of this polymerase allows repeated replication of input target cDNA in an isothermal exponential amplification. Our novel method overcomes two issues we originally identified using this polymerase. Amplification of linear DNA, such as cDNA, is more efficient in the center portion of the molecules so ends are under represented in the amplified product. Additionally, larger linear fragments are preferentially amplified over smaller fragments. We learned that ligation to form relatively large concatemers prior to amplification eliminated these issues. Double stranded cDNA was prepared from either total RNA or mRNA . The resulting fragments were then ligated to form a mix of both concatemers and circles. This material was then used as input for random primed amplification using phi29 DNA polymerase. Following a subsequent labeling step of the amplified cDNA, gene expression analysis was performed by microarray hybridization. Data will be presented to show results were consistent with those obtained using standard techniques requiring much larger amounts of RNA. We are investigating this novel amplification methodology to achieve gene expression analysis using smaller samples including mRNA amounts expected from a single cell.
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