A quantitative Hpall-PCR assay to measure methylation of DNA from a small number of cells

Abstract

Methylation-sensitive restriction enzymes and Southern blot analysis are commonly used to assay for DNA methylation, usually requiring DNA from about 105 cells. However sensitivity adequate for only a few hundred cells is needed in many cases, for example, the study of DNA methylation changes in germ cells or pre-implantation embryos. We earlier reported a sensitive PCR assay using primers that bracket a Hpall site; if the DNA is treated with HpaII prior to PCR, an amplified product is seen only from a methylated genomic template (1). We report here two improvements which make the assay quantitatively applicable to 100-300 cells, with as little as 10% DNA methylation detectable.We now include in each reaction an internal standard sharing primer binding sites with the genomic template, but distinguishable by size (2). The inset in Figure 1 shows the PCR products from the internal standard (band # 1), and from the genomic template (female blood DNA) with or without prior treatment with Hpall (band # 2). Also shown in Figure 1 are the densitometric tracings of these ethidium bromide-stained bands. Plotted in Figure 2 is the ratio of the internal standard band (I) to the genomic band (G) in an experiment in which the internal standard was varied from 80 to 240 molecules, while the genomic DNA (500 pg, approximately 100 molecules) was held constant. We find that female mouse spleen and blood DNA are methylated 52.5 2.3% and 50.6 i 3.5%, respectively, in agreement with previous data (1). To prepare DNA suitable for the HpaII-PCR assay, we used a guanidine HCl procedure (3), modified as described in the legend to Figure 1.

Early acquisition of homozygous deletions of p16/p19 during squamous cell carcinogenesis and genetic mosaicism in bladder cancer

“…In these samples, only two bladder cancer cases showed p16 promoter hypermethylation. This result is lower than our previous observations (14% vs 67%) of p16 exon 1 methylation in bladder TCCs (Gonzalez-Zulueta et al, 1995a) which was measured by the PCR based methylation analysis (Singer-Sam et al, 1990). We have, however, recently shown that methylation of the coding region in exon 1 does not inhibit the initiation of p16 transcription (Gonzalgo et al, 1998).…”

Dkk3, downregulated in cervical cancer, functions as a negative regulator of β‐catenin

“…27 We digested genomic DNAs either with MspI, which cleaves the CpG regardless of the methylation status, or with HpaII, which cleaves only unmethylated CpG. We then performed PCR with 3 pairs of primers designed for the promoter regions of the Dkk3 gene.…”

MIRA-SNuPE, a quantitative, multiplex method for measuring allele-specific DNA methylation

“…Restriction digestion with methylation sensitive enzymes using Southern blot 39 is an alternative method, and can be allele-specific if the region contains a suitable RFLP, but requires large amounts (about 10 μg) of genomic DNA. PCR detection of the methylation-sensitive site 40 requires less DNA but is not allele-specific. The drawback of these bisulfite-and restriction enzyme based methods is that they do not distinguish between 5mC and 5hmC.…”