Abstract
Rapid amplification of cDNA ends (RACE) is a widely used PCR-based method to identify the 5′ and 3′ ends of cDNA transcripts from partial cDNAs. While conceptually simple, this method often requires substantial optimization before accurate end identification is achieved. This is due in part to the anchoring of a universal primer to a cDNA or mRNA for PCR, which can lead to the generation of nonspecific amplification. Here, we describe an improvement of the original RACE method, circular RACE, which can be used to simultaneously identify both the 5′ and 3′ end of a target cDNA.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Salehi-Ashtiani K, Lin C, Hao T et al (2009) Large-scale RACE approach for proactive experimental definition of C. elegans ORFeome. Genome Res 19:2334–2342
Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002
Loh EY, Elliott JF, Cwirla S et al (1989) Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain. Science 243:217–220
Ohara O, Dorit RL, and Gilbert W (1989) One-sided polymerase chain reaction: the amplification of cDNA. Proc Natl Acad Sci USA 86:5673–5677
Schaefer BC (1995) Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem 227:255–273
Maruyama IN, Rakow TL, and Maruyama HI (1995) cRACE: a simple method for identification of the 5′ end of mRNAs. Nucleic Acids Res 23:3796–3797
Mandl CW, Heinz FX, Puchhammer-Stockl E et al (1991) Sequencing the termini of capped viral RNA by 5′-3′ ligation and PCR. Biotechniques 10:484, 486
Acknowledgment
We thank Andres Bendesky for critical reading of this protocol.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
McGrath, P.T. (2012). Characterizing cDNA Ends by Circular RACE. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press. https://doi.org/10.1007/978-1-61779-228-1_15
Download citation
DOI: https://doi.org/10.1007/978-1-61779-228-1_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-227-4
Online ISBN: 978-1-61779-228-1
eBook Packages: Springer Protocols