John M. S. Bartlett.pdf - Bio-Nica.info

106 Wang and Young
full-length cDNAs for a pineal-specific gene, PG23, were obtained in a single 5′-RACE
reaction because the antisense gene-specific primer used was derived from the common
3′ portion of the mRNAs (277 bp known sequence from differential display PCR or
DD-PCR, this chapter). This approach is suitable for cloning full-length cDNA quickly
based on a short sequence information from the 3′ end of mRNA, such as that obtained
by the DD-PCR technique (5) or expressed sequence tags (6). PG10.2 is a gene (8 kb
mRNA) expressed only in the pineal gland and the outer nuclear layer of the retina (7).
Two 4-kb cDNA fragments were obtained by 5′-RACE and 3′-RACE using primers
derived from only a 145-bp known sequence. Thus, this approach is suitable for cloning
full-length cDNA based on a short known sequence located anywhere on the mRNA,
whether derived from arbitrarily primed PCR (AP-PCR RNA fingerprinting technique
(8) or suppression subtractive hybridization (SSH) technology (Clontech, ref. 9). In
the case of PG10.2, a traditional 3′-RACE protocol was used to obtain the 3′ portion
of unknown sequence. Then, an anchored cDNA pool constructed using an antisense
gene-specific primer for reverse transcription was used to obtain the 5′ portion of
unknown sequence. This approach is especially reliable and useful for isolating fulllength
cDNAs for large transcripts (such as 8 kb for PG10.2) with low and restricted
expression. It may take only a few weeks to go from identification of differentially
expressed sequence tags (by DD-PCR, AP-PCR, or SSH technology) to full-length
cDNA by this long-template PCR-based RACE. In the following sections, both
5′-RACE and 3′-RACE protocols using the above model systems will be described.
The efficiency of described RACE approaches are very satisfactory for obtaining
full-length cDNAs quickly. The choice of method is dependent on the available
resources and relevant experience. For some genes, the use of more than one method
may be necessary. Another excellent approach for full-length cDNA cloning is using
biotin-tailed oligonucleotide probes to capture full-length cDNA clones from a wellconstructed
cDNA library (Invitrogen). A commercial kit called GeneTrapper cDNA
Positive Selection System has been built around this capture technology (Invitrogen).
2. Materials
1. RNA from source tissue (see Note 1): pineal gland and retina tissues were dissected
from Sprague–Dawley rats (male, 200–250 g, Taconic Farms). Total cellular RNA was
isolated using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions.
For preparation of double-stranded cDNA, poly (A)+ RNA was purified from 500 µg total
RNA using a Poly (A) Quik mRNA isolation kit (Stratagene, La Jolla, CA).
2. dNTP mixture: an aqueous solution of each dNTP (dGTP, dATP, dTTP, and dCTP) from
any reputable vendor of molecular biology reagents.
3. First-strand cDNA synthesis: SuperScript II (RNAse H-) reverse transcriptase (Invitrogen)
was used with manufacturer supplied 5 X reverse transcription buffer (see Note 2).
4. cDNA synthesis primer: this may be a universal oligo-dT primer (5′-CACTATAGGC
CATCGAGGCC(T) 20 MN-3′) for 5′-RACE and/or 3′-RACE (see Subheading 3.2.1.) or an
antisense gene-specific primer (5′-RACE only, see Subheading 3.4.2.) complementary to
the rare mRNA of interest or located upstream on a large transcript (see Note 3).
5. Second-strand cDNA synthesis: a 20× second-strand enzyme mixture, 5× second-strand
buffer and T4 DNA polymerase supplied in the Marathon cDNA amplification kit (Clontech
Laboratories, Inc.). Combining the following enzymes makes extra second-strand enzyme
mixture: Escherichia coli DNA polymerase I, E. coli DNA ligase, and E. coli RNAse H
(see Note 4).