Large-scale determination of the methylation status of retrotransposons in different tissues using a methylation tags approach

TY - JOUR

T1 - Large-scale determination of the methylation status of retrotransposons in different tissues using a methylation tags approach

AU - Khodosevich, Konstantin

AU - Lebedev, Yuri

AU - Sverdlov, Eugene D

PY - 2004/2/18

Y1 - 2004/2/18

N2 - A technique for simultaneous determination of the methylation status of numerous loci containing retroelements (REs) is reported. It is based on the observation that methylated and unmethylated areas in the genome are usually extended, and therefore the methylation of particular methyl-sensitive restriction endonuclease recognition sites might reflect the methylation status of DNA regions around them. The method includes dot-blot hybridization of repeat flanking sequences arrayed on a solid support with specifically amplified flanking regions of presumably unmethylated repeats. A multitude of flanking regions of REs adjacent to unmethylated restriction sites are amplified simultaneously, providing a complex hybridization probe. The technique thus allows the determination of the methylation status of restriction sites, which serve as tags of the methylation status of the surrounding regions. The validity of the technique was confirmed by various means, including bisulfite sequencing. The technique was successfully applied to the identification of methylation patterns of the regions surrounding 38 human-specific HERV-K(HML-2) long terminal repeats in cerebellum- and lymph node-derived genomic DNAs. The described technique can be readily adapted to the use of DNA microarray technology.

AB - A technique for simultaneous determination of the methylation status of numerous loci containing retroelements (REs) is reported. It is based on the observation that methylated and unmethylated areas in the genome are usually extended, and therefore the methylation of particular methyl-sensitive restriction endonuclease recognition sites might reflect the methylation status of DNA regions around them. The method includes dot-blot hybridization of repeat flanking sequences arrayed on a solid support with specifically amplified flanking regions of presumably unmethylated repeats. A multitude of flanking regions of REs adjacent to unmethylated restriction sites are amplified simultaneously, providing a complex hybridization probe. The technique thus allows the determination of the methylation status of restriction sites, which serve as tags of the methylation status of the surrounding regions. The validity of the technique was confirmed by various means, including bisulfite sequencing. The technique was successfully applied to the identification of methylation patterns of the regions surrounding 38 human-specific HERV-K(HML-2) long terminal repeats in cerebellum- and lymph node-derived genomic DNAs. The described technique can be readily adapted to the use of DNA microarray technology.

KW - Binding Sites/genetics

KW - Cerebellum/metabolism

KW - DNA Methylation

KW - DNA Primers/genetics

KW - Genetic Techniques/standards

KW - Genome, Human

KW - Humans

KW - Lymph Nodes/metabolism

KW - Nucleic Acid Hybridization/methods

KW - Reproducibility of Results

KW - Retroelements/genetics

KW - Terminal Repeat Sequences/genetics

U2 - 10.1093/nar/gnh035

DO - 10.1093/nar/gnh035

M3 - Journal article

C2 - 14973327

SN - 0305-1048

VL - 32

SP - e31

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 3

ER -