Production of in vitro amplified DNA pseudolibraries and high-throughput cDNA target amplification.

ABSTRACT

BACKGROUND: Many structural biology- and high-throughput laboratories experience the acquisition of multiple cDNAs from different sources as a rather time- and resource-consuming procedure. The techniques presented here solve these problems. RESULTS: An advanced target cDNA amplification procedure employing RNA- or cDNA-derived pseudolibraries circumvents the usual DNA transfection during library establishment. A small sample of reverse transcribed ss- or ds-cDNA or DNA from a pre-existing library is multiplied by in vitro rolling circle ramification amplification. The resulting cDNA pseudolibrary serves as a template for numerous highly efficient PCR amplifications and permits production and analysis of target cDNAs on an automated liquid handling workstation. CONCLUSION: The overall efficiency of the simple protocol collection approaches 100% for targets from libraries with low complexity such as Drosophila and yields >80% of amplicons up to 3 kb size in the case of human cDNA.