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

Rapid Amplification of cDNA Ends 111
Fig. 4. 5′- and 3′-RACE of PG10.2. An antisense gene-specific primer 2 (AGSP2 of PG10.2,
5′-GGCAGTTCATCCACACTCAGGTACCCAG-3′) and AP-1 primer amplified a faint but
sharp band of about 3.5 for 5′-RACE of PG10.2 (lane 1). Either AGSP2 (lane 2) or AP-1
(lane 3) alone failed to produce the band. A sense gene-specific primer (SGSP of PG10.2,
5′-GAGTGAGAAGCAAAGTGCAAATGCC-3′) and an anchor primer (5′-CCAAGCTATT-
TAGGTGACACTATAGGCCATCGAGGCC-3′, priming at the end of the newly synthesized
second-strand cDNA) amplified a band of 4 kb for the 3′-RACE (lane 4).
appeared even after a second round of PCR using nested primer set. This may due to nonoptimized
RACE PCR conditions or to an extremely low level of expression of the gene.
Other methods could be used to identify the correct RACE clones (see Note 12).
3.3. 3′-RACE Using Tailed Oligo-dT Primer for cDNA Synthesis
(see Note 9)
1. Northern analysis revealed a mRNA of 8.0 kb in size for PG10.2. Only 145 bp of sequence
information for this DD-PCR fragment was available to be used as template for RACE
primer design. After failing to find any positive cDNAs for PG10.2 in screens of two
cDNA libraries, we suspected that this was probably caused by the relatively far upstream
location of the 145 bp probe (Fig. 4 in ref. 7, underlined) and truncated clones in the cDNA
libraries we examined. In order to obtain this potentially large 3′ portion of unknown
sequence, we used a traditional 3′-RACE protocol outlined below to do 3′-RACE for
PG10.2. In general, the extended sequence information would provide a much broader
region for 5′-RACE primer design.