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dc.contributor.authorBower, Neil I.
dc.contributor.authorJohnston, Ian A.
dc.date.accessioned2011-12-05T16:33:59Z
dc.date.available2011-12-05T16:33:59Z
dc.date.issued2010-11
dc.identifier.citationBower , 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 . https://doi.org/10.1093/nar/gkq816en
dc.identifier.issn0305-1048
dc.identifier.otherPURE: 8682252
dc.identifier.otherPURE UUID: 2cdab890-5c49-4ec5-8f38-d65a65c66a92
dc.identifier.otherWOS: 000284952000003
dc.identifier.otherScopus: 78649831851
dc.identifier.otherORCID: /0000-0002-7796-5754/work/47136020
dc.identifier.urihttp://hdl.handle.net/10023/2090
dc.description.abstractAmplification 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.
dc.format.extent7
dc.language.isoeng
dc.relation.ispartofNucleic Acids Researchen
dc.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.en
dc.subjectUracil DNA Glycosylaseen
dc.subjectContaminationen
dc.subjectQH426 Geneticsen
dc.subject.lccQH426en
dc.titleTargeted rapid amplification of cDNA ends (T-RACE)-an improved RACE reaction through degradation of non-target sequencesen
dc.typeJournal articleen
dc.description.versionPublisher PDFen
dc.contributor.institutionUniversity of St Andrews.School of Biologyen
dc.contributor.institutionUniversity of St Andrews.Scottish Oceans Instituteen
dc.contributor.institutionUniversity of St Andrews.Centre for Research into Ecological & Environmental Modellingen
dc.contributor.institutionUniversity of St Andrews.Marine Alliance for Science & Technology Scotlanden
dc.identifier.doihttps://doi.org/10.1093/nar/gkq816
dc.description.statusPeer revieweden


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