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114 Wang and Young
3′-RACE outlined in Subheading 3.3. is more efficient because it relies on the sequence
introduced by the tail sequence 5′ of the oligo-dT sequence as the template for anchor
primer binding. The cDNA pool always provides more template for this kind 3′-RACE
than the one available from ligated adaptors, hence giving more robust 3′-RACE efficiency.
In general, the newly extended sequence information provides a larger region for design
of 5′-RACE primers. This is especially important if the available sequence information for
primer design is very limited. The gene-specific 5′-RACE protocol outlined in Subheading
3.4. is useful for obtaining full-length cDNAs of large transcripts or those expressed
at low levels. The cDNA produced by this protocol is only suitable for RACE PCR of
unknown sequence upstream of the gene-specific primer that was used for first-strand
cDNA synthesis.
10. Hot start PCR. The DNA polymerase is withheld from the reaction until the temperature
of reaction tube is above the annealing temperature. This hot-start PCR is used to improve
the specificity and sensitivity of the RACE PCR.
11. Co-solvent addition. For some primer/template systems such as GC-rich sequences, the
addition of glycerol and/or DMSO to a final concentration of 5% has been found to
enhance PCR yield and/or specificity.
12. The RACE PCR product may be cloned into pGEM-T vector even if agarose gel electrophoresis
fails to show a visible band(s). One dozen to one hundred colonies could be
screened for positive clones in colony hybridization using oligodeoxynucleotide probes
downstream from the RACE primer used for RACE PCR.
Acknowledgments
All experimental data presented in this chapter were performed in the former
Laboratory of Cell Biology, National Institute of Mental Health/National Institutes
of Health. We would like to acknowledge Dr. Michael J. Brownstein for invaluable
advice and generous support. We thank Dr. Herman E. Brockman for critical reading
of the manuscript.
References
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