Adaptor ligation-based polymerase chain reaction-mediated walking.
An improved method of adaptor ligation PCR was developed for isolation of unknown sequences flanking a known DNA sequence. It was determined that the specificity of the adaptor ligation-based walking technique could be significantly enhanced by using uniquely blocked adaptors along with removal of unligated genomic DNA by exonuclease III digestion. This technique was utilized to isolate three novel promoter regions from three different Zea mays (maize) peroxidase genes. Sequences encoding a putative maize 6-phosphogluconate dehydrogenase gene were also isolated and confirmed by sequence analysis. The described improvements could be applied to other existing adaptor ligation-based PCR walking techniques.
Padegimas
L S
LS
Department of Plant and Soil Sciences, Mississippi State University, Mississippi State, Mississippi 39762, USA.
Reichert
N A
NA
D001483:Base Sequence; D017931:DNA Primers; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D005090:Exodeoxyribonucleases; D017343:Genes, Plant; D007202:Indicators and Reagents; D009690:Nucleic Acid Conformation; D010544:Peroxidases / Q000235:genetics*; D010734:Phosphogluconate Dehydrogenase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D017931:DNA Primers; D018744:DNA, Plant; D007202:Indicators and Reagents; D010734:Phosphogluconate Dehydrogenase; D010544:Peroxidases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III