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.

Epigenetic changes in virus-associated human cancers

“…The other non-array based method like RLGS [80][81][82] uses the methylation-sensitive enzyme like NotI to create a genome-wide methylation differences between the tumor and non-tumor parts. These DNAs were then labeled with isotope, and profiled onto two reproducible two-dimensional gels.…”

Suppression of the protein tyrosine phosphatase receptor type O gene (PTPRO) by methylation in hepatocellular carcinomas

“…NotI sites are located predominantly in the CpG islands and becomes resistant to the enzyme upon methylation of the cytosine within CpG (Hayashizaki et al, 1993). We used this technique to explore genome-wide changes in the methylation profile during folate-induced hepatocarcinogenesis in rats.…”

Inheritance of DNA Methylation in Plant Genome

“…Restriction Landmark Genome Scanning (RLGS) [Hayashizaki et al 1993] is a unique quantitative approach well suited for simultaneous assay of methylation status [Costello et al, 2002]. The other genome wide methylation analyze methods viz., Tiling microarrays and methylation-sensitive amplification polymorphism (MSAP) [Reyna-Lopez et al 1997, Xiong et al 1999 are the comprehensive or easily applied method, respectively, but RLGS excels these methods because of its reduced cost and short span of experimental time (3 days).…”

“…Thousands of loci with high reproducibility can be detected as spots on the 2-D pattern in this method. A lot of methylated sites can be analyzed using the conventional RLGS method because the recognition site (GCGGCCGC) of the first cutter NotI [Hayashizaki et al 1993, Watanabe et al 1995, which is a methylation sensitive restriction enzyme (Fig. 1A), is often located in the CpG islands [Bird 1992].…”