Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots.
Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, labor-intensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.
Laprise
Shari L
SL
Math/Science Division, Babson College, Forest Street, Babson Park, MA 02457, USA. slaprise@babson.edu
Gray
Mark R
MR
D044503:5-Methylcytosine / Q000378:metabolism; D000818:Animals; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005176:Factor XIII / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000235:genetics
D018343:Receptors, Adrenergic, beta-2; D011965:Receptors, Glucocorticoid; D044503:5-Methylcytosine; D004247:DNA; D005176:Factor XIII; D015246:Deoxyribonuclease EcoRI; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; C040158:GTAC-specific type II deoxyribonucleases