Cloning and direct sequencing of plant promoters using primer-adapter mediated PCR on DNA coupled to a magnetic solid phase.
A method that allows amplification and direct sequencing or cloning of an unknown DNA segment flanked by a known sequence is described using barley genomic DNA. The method avoids the step of circularization necessary for inverse PCR by ligation of primer-adapters to restricted genomic DNA. Specificity is achieved in the first amplification step; linear PCR with a biotinylated primer complementary to the known flanking sequence (primer 1-B) produces a single-stranded product that is purified employing streptavidin-coated magnetic beads. After this step, which removes genomic DNA, two rounds of exponential PCR are performed, first with the adapter-primer and primer 1 and second with primer 1 substituted by a nested primer 2. If the second primer is biotinylated, the product can be sequenced directly using solid-phase sequencing. We have employed this method to sequence directly and to clone the promoters of two late embryogenesis-abundant (Lea) genes (B19.4 and B19.3) from barley. Lea B19.4 and B19.3 encode putative desiccation-protective proteins that act in the final stages of embryogenesis and have previously been cloned as cDNAs. We demonstrate here that their proximal promoter regions are very similar (80% identity) and that both contain putative abscisic acid-responsive elements.
Espelund
M
M
University of Oslo.
Jakobsen
K S
KS
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D001467:Hordeum / Q000235:genetics; D008280:Magnetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D013698:Templates, Genetic
D004247:DNA