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Rapid Amplification of cDNA Ends 105
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Rapid Amplification of cDNA Ends
Xin Wang and W. Scott Young III
1. Introduction
The identification and isolation of full-length cDNAs can be a frustrating and timeconsuming
experience, especially for genes with a low abundance of expression or with
large transcripts. Traditionally, full-length cDNAs are obtained from cDNA libraries
by hybridization with radioisotope-labeled probes. This labor-intensive and tedious
procedure often produces incomplete sequences and sometimes includes intronic
sequence. To obtain full-length cDNAs, investigators had to rescreen libraries with
larger numbers of clones (or upstream probes), not always successfully. The combination
of rapid amplification of cDNA ends (RACE) and long-distance polymerase chain
reaction (PCR) with high fidelity makes it possible to obtain full-length cDNAs quickly
without constructing or screening a cDNA library.
The principle of RACE is simple and elegant: An anchor sequence is added to the
end of the cDNA to be used as PCR primer binding template. A universal primer
complementary to the added anchor template is coupled with a gene-specific primer
(based on a single short known sequence within the mRNA of interest) in a PCR to
amplify regions with unknown sequence. Several strategies have been developed to
isolate full-length cDNA using this anchored PCR technology, each using a unique way
to add the anchor sequence to the end of the cDNA. In the first generation of RACE,
homopolymeric tails (G or A) are added to 3′ end of cDNA to be used as an anchor
sequence using the enzyme terminal deoxynucleotidyl transferase (1). The second
generation of RACE technique is based on the ability of T4 RNA ligase to ligate
a single-stranded anchor sequence to the 3′ end of the first-strand cDNA (2). Both
methods are difficult to optimize because of inefficient enzymatic reactions. The third
generation of RACE uses T4 DNA ligase to add a double-stranded anchor sequence to
both ends of double-stranded cDNAs (3), thus the resulting anchored cDNAs are suitable
for both 5′- and 3′-RACE. A commercial kit called Marathon cDNA amplification
kit has been built around this approach (Clontech Laboratories, Inc.).
In this chapter, the application of this third-generation RACE method to the isolation
of several pineal-specific cDNAs ranging from 1.4 to 8.0 kb in size is outlined. In
one case, a full-length 2.0-kb cDNA for a pineal-specific cDNA, PG25, was obtained
by 5′-RACE using gene-specific primers based on 260 base pairs of known sequence
located in the 3′ terminus of the mRNA (4). In another case, two different versions of
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
105