title: Limiting Dilution Bisulfite Pyrosequencing®: A Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells.
abstract: Bisulfite-based methods for DNA methylation analysis of small amounts of DNA from a limited number of cells are technologically challenging. Degradation of genomic DNA by bisulfite treatment, contamination with foreign DNA, and biases in the amplification of individual DNA molecules can generate results, which are not representative of the starting sample. Limiting dilution (LD) bisulfite Pyrosequencing(®) (BSP) is a relatively simple technique to circumvent these problems. The bisulfite-treated DNA of a single or a few cells is diluted to an extent, that only a single DNA target molecule is present in the reaction. Then each individual DNA molecule in the starting sample is separately amplified and analyzed by Pyrosequencing. This allows the detection of rare alleles that are easily masked when pools of DNA target molecules are analyzed. Amplicons containing a heterozygous single nucleotide polymorphism (SNP) allow one to delineate the parental origin of the recovered molecules in addition to their methylation status. The number of cells (DNA target molecules) in the starting sample determines the dilution level and the number of reactions that have to be performed. LD-BSP allows methylation analysis of small cell pools (i.e., 5-10 microdissected cells) and even individual cells. The primers and PCR conditions described here have been successfully employed to analyze the methylation status of up to eight target genes in individual 2-16 cell embryos, germinal vesicle (GV) oocytes, and haploid sperms.
authors.affiliation: Institute of Human Genetics, Julius-Maximilians University Würzburg, Würzburg, 97074, Germany.
authors.forename: Nady El
authors.initials: NE
authors.lastname: Hajj
authors.forename: Juliane
authors.initials: J
authors.lastname: Kuhtz
authors.forename: Thomas
authors.initials: T
authors.lastname: Haaf
mesh_terms: D000818:Animals; D016477:Artifacts; D002417:Cattle; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D019175:DNA Methylation / Q000187:drug effects*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D051379:Mice; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite