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

Rapid Amplification of cDNA Ends 109
Fig. 2. Schematic representation of 5′- and 3′-RACE for the 8.0 kb cDNA of PG10.2. (A)
The 145-bp sequence of PG10.2 is located in the middle of the 8-kb mRNA. (B) 3′-RACE.
SGSP coupled with the anchor primer (complementary to the tail sequence of oligo-dT cDNA
synthesis primer) for 3′-RACE. (C) 5′-RACE. AGSP2 coupled with the AP-1 anchor primer
for 5′-RACE.
buffer, and 1 µL of T4 DNA ligase were incubated at 16°C overnight. The mixture was
heated at 70°C to inactivate the ligase.
5. Primer design for PG23 5′-RACE: A short known sequence located at the 3′ end region
of the gene is required for designing antisense gene-specific primers (AGSP) and a
nested antisense gene-specific primer (NAGSP) for 5′-RACE. In the case of PG23, AGSP
(5′- GGAACAGTCTGAGCTCTAAGCTAGG-3′) and NAGSP (5′-TCTAGAAGGATA-
AGTTCACGAGGGCC-3′) were designed based on the 277-bp sequence information just
upstream of the poly (A) tail as shown in Fig. 3.
6. PCR was performed in a 50-µL reaction containing the following components: 5 µL of
10× PCR buffer 1, 5 µL of the 1:100 diluted adaptor-ligated cDNAs, 1 µL of 10 mM
dNTP, 1 µL of 10 µM AP-1 linker primer, 1 µL of 10 µM GSP, and 0.75 µL of DNA
polymerase mixture (added last). The DNA polymerase mixture was added to the reaction
after denaturation at 94°C for 1 min to reduce nonspecific amplification (see Note 10).