Amplification and cloning of sugarcane sucrose synthase cDNA by anchored PCR

We have used a strategy based on the polymerase chain reaction (PCR) to amplify and construct full-length sucrose synthase (SS) cDNA of sugarcane. Two SS-specific internal primers were synthesized based on their complementarity to published consensus sequences of the SS gene of maize and wheat. Amplification of full-length cDNA was achieved by an anchored PCR method utilizing primers which extend to 5' and 3' ends of specific cDNA. In the first step, a homopolymeric oligo(dC) tail was added to the 3' end of single-stranded cDNAs. The two SS cDNAs were amplified, one with a 5' end (SSp1) and the other with a 3' end (SSp2) using one internal SS primer and the other anchored end primer. Finally, overlapping fragments were identified by restriction mapping, and the non-overlapping fragments were excised and religated to reconstruct full-length cDNA. Partial sequences of the reconstructed cDNAs (SS-5' and SS-3') were compared with the published SS sequences to confirm that the amplified DNA was a copy of the SS transcript.