Rolling circle amplification of genomic templates for inverse PCR (RCA–GIP): a method for 5′- and 3′-genome walking without anchoring

Abstract

We have devised an improved method of genome walking, named rolling circle amplification of genomic templates for Inverse PCR (RCA-GIP). The method is based on the generation of circular genomic DNA fragments, followed by rolling circle amplification of the circular genomic DNA using phi29 DNA polymerase without need for attachment of anchor sequences. In this way from the circular genomic DNA fragments, after RCA amplification, a large amount of linear concatemers is generated suitable for Inverse PCR template that can be amplified, sequenced or cloned allowing the isolation of the 3'- and 5'- of unknown ends of genomic sequences. To prove the concept of the proposed methodology, we used this procedure to isolate the promoter regions from different species. Herein as an example we present the isolation of four promoter regions from Crocus sativus, a crop cultivated for saffron production.