Genomic sequencing and methylation analysis by ligation mediated PCR.
Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.
Pfeifer
G P
GP
Molecular Biology Section, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Steigerwald
S D
SD
Mueller
P R
PR
Wold
B
B
Riggs
A D
AD
D044503:5-Methylcytosine; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D003596:Cytosine; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D014960:X Chromosome
D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; D010735:Phosphoglycerate Kinase