Restriction site extension PCR: a novel method for high-throughput characterization of tagged DNA fragments and genome walking.

ABSTRACT:

Background

Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner.

Methodology/principal findings

We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR) to efficiently conduct large-scale isolation o

SUBMITTER: Ji J 

PROVIDER: S-EPMC2868030 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature