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BMC biotechnology

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


PMID 17565673

Abstract

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. 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. 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.

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