Reference "Amplification and cloning of sugarcane sucrose synthase cDNA by anchored PCR"

Reference ID

8979

Title

Amplification and cloning of sugarcane sucrose synthase cDNA by anchored PCR

Source

PCR Methods Appl, 1992, vol. 2, pp. 70-75

Authors (3)

Kumar-A-S	Moore-P-H
Maretzki-A

Abstract

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.