restriction site extension pcr a novel method for high-throughput characterization of tagged dna fragments and genome walking限制网站延伸pcr方法标记dna片段和基因组高通量表征的散步.pdfVIP

Restriction Site Extension PCR: A Novel Method for High- Throughput Characterization of Tagged DNA Fragments and Genome Walking ¤ Jiabing Ji* , Janet Braam* Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America 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 of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 39 overhang of the endonuclease (KpnI, NsiI, PstI, or SacI) restricted DNA fragments, extends the 39 end of the DNA fragments in the first cycle of the primary RSE- PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR), touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis . All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demon