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Please use this identifier to cite or link to this item: http://hdl.handle.net/10023/2090
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Title: Targeted rapid amplification of cDNA ends (T-RACE)-an improved RACE reaction through degradation of non-target sequences
Authors: Bower, Neil I.
Johnston, Ian A.
Keywords: Uracil DNA Glycosylase
Contamination
QH426 Genetics
Issue Date: Nov-2010
Citation: Bower , N I & Johnston , I A 2010 , ' Targeted rapid amplification of cDNA ends (T-RACE)-an improved RACE reaction through degradation of non-target sequences ' Nucleic Acids Research , vol 38 , no. 21 , e194 .
Abstract: Amplification of the 5' ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3' end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3' or 5' end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3' or 5' T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3' and 5' ends of numerous cDNAs from a single cDNA synthesis reaction.
Version: Publisher PDF
Status: Peer reviewed
URI: http://hdl.handle.net/10023/2090
DOI: http://dx.doi.org/10.1093/nar/gkq816
ISSN: 0305-1048
Type: Journal article
Rights: © The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Appears in Collections:University of St Andrews Research
Biology Research
Centre for Research into Ecological & Environmental Modelling (CREEM) Research
Scottish Oceans Institute Research



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