Figure 1

Random directional cDNA synthesis. A. The first strand cDNA primer (DRP1) contains: a 5' phosphorylated buffer sequence devoid of A bases (1), an AscI site (2), a 5 nt random sequence (3), and a 3' T residue (4). By design, priming should begin only at an A in the RNA template. B. The non-phosphorylated lone-linker LL1 consists of the the two complimentary oligonucleotides LL1F and LL1R. It has one blunt end and one non-adhesive staggered end. LL1 can therefore ligate only to one strand of the cDNAs and in only one orientation. The remaining nick in the second strand is removed by preincubating the cDNAs before the first PCR reaction at 72°C for one minute to strip off the non-ligated strand of the linker and regenerate the sequence by extension from the 3' end of the cDNA (lower grey).