Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

Abstract

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, laborintensive 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.

Effects of non-CpG site methylation on DNA thermal stability: a fluorescence study

“…( 19 ) recently proposed that the differential stability of methylated versus non-methylated duplexes might be exploited to probe the amount of methylation in both specific genes and entire genomes in a smart and sensitive way. Indeed, substitution of methylcytosine for cytosine in the context of CpG repeats has been previously shown to enhance the double-strand stability during both thermal ( 20 – 22 ) and chemical denaturation ( 20 , 21 , 23 ). Notably, the results of Diede et al .…”

Differential Methylation as a Cause of Allele Dropout at the ImprintedGNASLocus

“…CpG methylation has been suggested to alter the DNA secondary structure and melting properties (Laprise and Gray, 2007), which may impair PCR amplification (Kholod et al, 2007). However, to our knowledge, the interference of the differential methylation at imprinted regions in PCR amplification has never been reported.…”

False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

“…Methylation status can alter the DNA secondary structure and melting properties 10 . Amplification is not prevented by methylation, but it is rather driven preferentially toward the unmethylated allele when the methylation status of the two alleles is distinct 11 .…”

“…T a = primer annealing temperature; PCR = polymerase chain reaction. Preferential engagement of the polymerase to a specific allele can have multiple causes, like differences in GC content between alleles, heterozygous variants in the primer region, methylation status or altered folding [8][9][10][11] . A different variant in the primer annealing site was excluded as a cause due to equivalent results when using different primer sets.…”