Restriction landmark genomic scanning method and its various applications

Abstract

We have developed a new genome scanning method (restriction landmark genomic scanning (RLGS), based on the new concept of using restriction enzyme sites as landmarks. RLGS employs direct end labeling of the genomic DNA digested with a restriction enzyme and two-dimensional electrophoresis with high-resolution. Its advantages are: (i) high-speed scanning ability, allowing simultaneous scanning of thousands of restriction landmarks; (ii) extension of the scanning field using different kinds of landmarks in an additional series of electrophoresis; (iii) application to any type of organism because of direct-labeling of restriction enzyme sites and no hybridization procedure; and (iv) reflection of the copy number of the restriction landmark by the spot intensity which enables distinction of haploid and diploid genomic DNAs. The RLGS method has various applications because it can be used to scan for physical genomic DNA states, such as amplification, deletion and methylation. The copy number of the locus of a restriction landmark can be estimated by the spot intensity to find either an amplified or deleted region. The methylation state of genomic DNA can also be discovered by use of a methylation-sensitive restriction enzyme sites as a restriction landmark (restriction landmark genomic scanning for screening methylated sites, RLGS-M). This article introduces the basic principle of RLGS and its applications to the analysis of cancer, mouse mutant DNAs and tissue-specific methylation, showing the usefulness of RLGS for a variety of biological fields.