Restriction landmark genome scanning method using isoschizomers (MspI/HpaII) for DNA methylation analysis

Abstract

Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, MspI and HpaII, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with MspI and HpaII. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C(5m)CGG sites. Further analyses revealed an additional methylated site of NotI. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.

Inheritance of DNA Methylation in Plant Genome

“…This software can be utilized for the RLGS analysis of the organisms for those the whole genome sequence is available. One among such organism is Rice which enables the utilization of "in silico RLGS" analysis [Takamiya et al 2006] RLGS is a high-speed genome scanning system. It employs direct end-labeling of the genomic DNA digested with rare-cutting restriction enzyme such as NotI, followed by highresolution two-dimensional (2-D) electrophoresis.…”

“…In the Takamiya et al 2006, the methylated status was compared among 3 ecotypes of Arabidopsis using the RLGS method. Methylation at a total of 17 sites (NotI: 9 sites and HpaII: 8 sites) was detected in the 3 ecotypes.…”

Application of RLGS Method for Detection of Alteration in Tissue Cultured Plants

“…We employed the improved spot cloning method 12 based on conventional methods 3,4 . Briefly, the genomic DNA (0.1 µg) was digested by NotI and HpaII at 37ºC for 1 h. Then, the sample was mixed with DNA fragments labeled at the NotI sites and digested with HpaII.…”

“…Briefly, the genomic DNA (0.1 µg) was digested by NotI and HpaII at 37ºC for 1 h. Then, the sample was mixed with DNA fragments labeled at the NotI sites and digested with HpaII. Thereafter, 2-D electrophoresis was carried out as described by Okamoto et al 11 and Takamiya et al 12 . The target RLGS spots were punched out from the dried gel and soaked in Tris-EDTA (TE) buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).…”

“…We carried out RLGS with restriction enzyme combination, NotI-HpaII-BamHI (NEB, Beverly, MA, USA), described in Okamoto et al 11 and Takamiya et al 12 .…”