title: Ligation-mediated PCR improves the sensitivity of methylation analysis by restriction enzymes and detection of specific DNA strand breaks.
abstract: DNA methylation at specific sites is most frequently studied by use of methylation-sensitive restriction endonucleases and Southern blotting. We report here that the sensitivity of this method can be increased several-hundred-fold by applying a ligation-mediated polymerase chain reaction (LM-PCR) procedure following enzyme treatment. DNA is cleaved simultaneously with two restriction enzymes, one sensitive and one insensitive to methylation. After cleavage, a gene-specific oligonucleotide primer is used for primer extension, followed by linker ligation and then conventional PCR. Using this technique, we demonstrate that DNA from 100 cells (about 0.6 ng) can be prepared and qualitatively analyzed for methylation at sites in an X-linked CpG island, and 50 ng of DNA can be analyzed quantitatively. A site 23 bp downstream of the major transcription start site of human phosphoglycerate kinase-1 (PGK-1) is 52 +/- 7 percent methylated in DNA from female blood and greater than 98 percent unmethylated in DNA from male blood or HeLa cells. This method detects quantitatively specific breaks in either double stranded or single stranded DNA. Thus new assays for enzymes and DNA structure can be devised.
authors.affiliation: Department of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010.
authors.forename: S D
authors.initials: SD
authors.lastname: Steigerwald
authors.forename: G P
authors.initials: GP
authors.lastname: Pfeifer
authors.forename: A D
authors.initials: AD
authors.lastname: Riggs
mesh_terms: D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004249:DNA Damage; D004262:DNA Restriction Enzymes; D006801:Humans; D006822:Hybrid Cells; D008745:Methylation; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping; D014960:X Chromosome
chemical_list: D015345:Oligonucleotide Probes; D004247:DNA; D004262:DNA Restriction Enzymes