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Short communication
Abstract
A PCR-based method for the synthesis of high quality cDNA is described. This method combines the reverse transcriptase template switching
with the assisted recombination cloning. It enables 50 end enrichment, cDNA synthesis from limiting starting material, elimination of digestion and
subcloning steps and substantial time and cost savings.
# 2007 Elsevier B.V. All rights reserved.
Keywords: cDNA library; Recombination; Cloning; Reverse transcription
* Corresponding author.
E-mail address: andreia.figueiredo@icat.fc.ul.pt (A. Figueiredo).
1
Laboratory of Molecular BiologyICAM (Institute for Mediterranean
Agrarian Sciences) Evora University, Polo da Mitra, 7002-554 Evora, Portugal.
1389-0344/$ see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bioeng.2007.05.004
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Fig. 1. Schematic representation of the cDNA library construction method reported. (A) The first strand synthesis is achieved in two steps: first, the attB2-oligo(dT)
primer hybridizes to the mRNA poly(A)+ tail and then, the Superscript II reverse transcriptase synthesizes the first strand of the cDNA, using the poly(A)+ RNA as a
template, adding a (dC) tailing where the attB1 primer hybridizes. (B) The resulting cDNA was treated with RNAseH. (C) In a PCR reaction using the first strand
cDNA as a template and the 50 attB1 and attB2-oligo(dT) as primers, the second strand is synthesized and the attB recombination sites are created. (D) The cDNA
flanked by the attB1 and attB2 recombination sites is transferred to the entry vector pDNORTM221 by the Gateway BP recombination reaction creating an entry
library and a by-product.
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