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

Rapid Amplification of cDNA Ends 107
6. Adaptor (anchor sequence) ligation: A specially designed partially double-stranded adaptor
supplied in the Marathon cDNA amplification kit (Clontech Laboratories, Inc.).
7. Oligonucleotide primers: gene-specific primers should be about 25 nucleotides long and
around 50% guanine–cytosine. Primers with a melting temperature between 65 and 70°C
give sufficient binding specificity (see Note 5). For some difficult genes, primers with
70°C melting temperature or higher should be used in a touchdown PCR (see Note 6).
Anchor primers (AP) complementary to the adaptor sequence (for 5′-RACE or 3′-RACE) or to
the tailing sequence on the cDNA synthesis oligo-dT primer are coupled with GSP to amplify
the unknown sequences flanked by the paired primer set. AP-1 (5′-CCATCCTAATACGACT
CACTATAGGGC-3′) and AP-2 (nested within the AP-1, 5′-ACTCACTATAGGGCTC
GAGCGGC-3′) are supplied in Clontech’s Marathon cDNA Amplification Kit.
8. PCR machine: all experimental data presented in this paper were carried out on a
thermocycler from MJ Research, Inc (see Note 7).
9. DNA Polymerase: Expand PCR system (Boehringer-Mannheim). There are three buffer
systems supplied by the manufacturer. Buffer 1 is sufficient for RACE PCR of expected
product size of less than 10 kb.
10. PCR fragment purification: QIAEX II from QIAGEN Bioscience Corporation.
11. TA cloning: pGEM-T vector system (Promega Corporation).
12. Other general molecular laboratory equipment and reagents.
13. All reagents, including enzymes, should be mixed briefly immediately before use.
3. Methods
3.1. The Relative Abundance of the Transcript in the Source Tissue
1. A good understanding of expression profile is essential for the successful isolation of the
full-length cDNA of interest. The efficiency of RACE PCR amplification largely depends
on the relative abundance of the mRNA of interest in the poly (A)+ RNA sample extracted
from the target tissue. RACE PCR should be performed on the tissue where the expression
is most abundant. The higher the copy number of the mRNA in the cDNA pool, the better
chance the full-length cDNA can be amplified to a critical mass visible on agarose gel
(see Note 8).
2. PG23 is a pineal-specific gene identified using DD-PCR (unpublished data). In this case,
we had no choice but to use pineal as our target tissue for 5′-RACE. Northern blot analysis
revealed a mRNA doublet about 2.0 kb and 2.4 kb for PG23 (Fig. 1A, lane 1). The size of
mRNA serves as a useful guide in identifying the correct RACE bands (Fig. 1B, lanes 1
and 2). In a similar fashion, another 2.0-kb full-length pineal-specific cDNA (PG25) was
obtained using the RACE protocol outlined for PG23 (Fig. 3B in ref. 4).
3. PG10.2 is a gene (8 kb mRNA) expressed only in the pineal gland and the outer nuclear
layer of the retina (7). In this case, retina was used as target tissue source because PG10.2
has a much higher expression level in retina than in pineal even though it is only expressed
in the outer nuclear layer of the retina (Fig. 3 in ref. 7). This report will describe the
application of a traditional 3′-RACE (see Subheading 3.3.) and a gene-specific 5′-RACE
(see Subheading 3.4.) to the isolation of the large transcript of PG10.2 (8.0 kb). A general
strategy is schematically depicted in Fig. 2.
3.2. 5′-RACE Using a Universal Oligo-dT Primer for cDNA Synthesis
(see Note 9)
1. First-strand cDNA synthesis: 1 µg of high-quality poly (A)+ mRNA isolated from fresh
rat pineal was reverse-transcribed to first-strand cDNA in a 10-µL reaction containing
1 µL of 10× first-strand synthesis buffer, 1 µL of 0.1 M dithiothreitol, 1 µL of 10 mM