Figure 1.

The RNA-ligase-mediated 5′-RACE procedure to clone the 5′ terminus of a viral RNA genome. Viral RNA is converted to cDNA with random primers by reverse transcription. A phosphorylated synthetic oligodeoxynucleotide adapter is ligated to the 3′ end of cDNA by using T4 RNA ligase, and then two rounds of PCR amplification are performed. The first-round PCR is done with the adapter primer (AP-1), complementary to the 3′ end of the adapter, and the viral-specific primer 1 (VS-1). The second-round nested PCR is done with the other adapter primer (AP-2), complementary to the 5′ portion of the adapter, and the viral-specific primer 2 (VS-2). The PCR products are subjected to cloning and then sequencing. The RNA molecule is represented as a wavy line, cDNA molecules are straight lines, adapters are thick lines, and primers are short arrows.