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.
Department of Genetics and Pathology, Hawaiian Sugar Planters' Association, Aiea 96701.
A S
AS
Kumar
P H
PH
Moore
A
A
Maretzki
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics*; D017343:Genes, Plant; D005964:Glucosyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins / Q000235:genetics*; D010945:Plants, Edible / Q000235:genetics*; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification
D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D012333:RNA, Messenger; D011061:Poly A; D004247:DNA; D005964:Glucosyltransferases; C022699:sucrose synthase