Application of the RLGS Method to Large-Size Genomes Using a Restriction Trapper

Abstract

We developed a method for producing restriction landmark genomic scanning (RLGS) profiles of largesize genomes, such as those of higher plants or amphibians using a restriction trapper. Use of the conventional RLGS method is limited to genomes smaller than 3 x 10 9 bp, because the larger genomic DNAs, especially those of more than 1 x 10 10 bp, produce high background due to incorporation of radioactivity at non-specifically damaged sites. Our new method reduces the background levels by reducing genome complexity to 1/200-1/300 using a purification step to enrich DNA fragments carrying specific restriction landmarks at their ends using a restriction trapper. This step makes it possible to obtain RLGS patterns of larger genomes. Our paper describes the practical application for the RLGS method using a restriction trapper with the pine tree genome (3 x 10 10 bp/haploid genome; Pinus koraiensis Sieb. et Zucc.) as an example.