Evaluation of PCR-based methods for isolating flanking regions of genes.
Several polymerase chain reaction (PCR)-based methods are available for isolation of unknown genomic fragments. In the present study, a comparative evaluation of a few methods of ligation-mediated PCR methods and a ligation-independent one were made by isolating promoter fragment for N-methyltransferase gene involved in the caffeine biosynthetic pathway of Coffea canephora. The benefits of tertiary PCR and the effects of a 4-base cutting restriction endonuclease on the size of the PCR products obtained were demonstrated in one of the ligation-mediated PCR methods. The methods adopted in this study differed in the sizes of the 5'-flanking regions obtained. The efficiencies of various methods used reflect the inherent limitations of the PCR-based methods for isolation of unknown flanking regions.
Satyanarayana
K V
KV
Plant Cell Biotechnology Department, Central Food Technological Research Institute (CFTRI), Mysore, Karnataka 575020, India.
Chandrashekar
A
A
Ravishankar
G A
GA
D024506:5' Flanking Region; D001709:Biotechnology / Q000379:methods*; D016386:Chromosome Walking; D040503:Coffea / Q000235:genetics / Q000378:metabolism; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D017343:Genes, Plant; D008780:Methyltransferases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic
D018744:DNA, Plant; D008780:Methyltransferases