Methylation analysis using bisulfite genomic sequencing: application to small numbers of intact cells.

This report describes the application of bisulfite genomic sequencing to samples consisting of between 50 and 200 intact cells. Bisulfite genomic sequencing, originally described by Frommer et al. (5), allows the determination of methylation status of each cytosine residue in a defined sequence for individual strands of genomic DNA. This technique relies on the ability of bisulfite to specifically convert unmethylated cytosine residues, but not 5methylcytosine, to uracil by deamination (6,7,10,12). Bisulfite-modified DNA is then polymerase chain reaction (PCR)-amplified to yield products in which uracil residues have been amplified as thymine and 5-methylcytosine residues only have been amplified as cytosine. The methylation pattern of a target sequence is revealed when PCR products are sequenced directly, to reveal an average methylation pattern for the population of DNA molecules, or cloned and sequenced to reveal the methylation pattern of an individual allele. Published variations of the bisulfite genomic sequencing protocol require microgram amounts of restriction enzyme-digested genomic DNA as starting material (2,4,5,9) or dilutions to picogram amounts of previously digested genomic DNA (2). In this report, we describe a method that allows the bisulfite genomic sequencing technique to be used for the methylation analysis of genes when starting material consists of between 50 and 200 intact cells. This method will allow researchers to investigate the role of methylation in the control of gene expression in germ cells, embryogenesis, inherited diseases and cancers, when samples are limiting. However, since it may be possible to amplify from a single allele, minimizing cell numbers too drastically will give methylation profiles that are not representative of the population of cells. This is especially the case since no method of DNA purification, restriction enzyme digestion or PCR amplification can be 100% efficient. Thus, overall experimental design should allow for this by including as many cells per sample as possible, and multiple individual samples must be assayed. We have used this method in our laboratory to generate comprehensive methylation profiles of the Xist gene in mouse germ cells and during preimplantation development in order to study the role of methylation in the genomic imprinting of this gene. F1 generation embryos derived from evolutionary distinct strains of mice were used in these experiments so that polymorphisms could be used to identify the parental origin of individual alleles. In the first step of the procedure, genomic DNA is prepared from cells treated with a lysis buffer containing proteinase K and guanidine hydrochloride (8). Genomic DNA is then recovered from the lysate by ethanol precipitation in the presence of glycogen, which acts as a carrier for low concentrations of DNA. Bisulfite modification of isolated genomic DNA is then performed essentially as described by Clark et al. (2) with variations introduced to increase the sensitivity of the procedure for small amounts of starting material. As bisulfite induced deamination of cytosine residues is only efficient for single-stranded DNA (6,11), the genomic DNA is first denatured by restriction enzyme digestion followed by alkali denaturation. The efficiency of the procedure was increased by including plasmid DNA in the restriction enzyme digest and by increasing the stringency of denaturation by raising the reaction temperature from 37°C to 75°C. Raizis et al. (9) report that shearing genomic DNA before alkali denaturation is an unnecessary step; however, in our experience this step was found to be essential (data not shown). Prolonged bisulfite treatment causes degradation of DNA as a result of depurination (9). For this reason, the kinetics of the bisulfite modification reaction were maximized by using 4.8 M bisulfite, which allowed reaction time to be reduced to 4 h. For PCR amplification, we recommend using touchdown PCR (3) for amplifying bisulfite-modified DNA in picogram amounts. There are at least two advantages of using touchdown PCR over the traditional PCR approach. First, it can avoid the amplification of nonspecific products, which is a possibility with the T-rich forward primers or A-rich reverse primers used to amplify bisulfite-modified DNA. Second, it can help overcome the problem of arriving at a suitable empirically derived annealing temperature for these primers without time-consuming optimization procedures. The optimized protocol for bisulfite genomic sequencing starting with small samples of intact cells is as follows: Cells in a volume of phosphatebuffered saline (PBS) + 0.1% bovine