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. 2000 Apr 15;28(8):E32.
doi: 10.1093/nar/28.8.e32.

MethyLight: A High-Throughput Assay to Measure DNA Methylation

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Free PMC article

MethyLight: A High-Throughput Assay to Measure DNA Methylation

C A Eads et al. Nucleic Acids Res. .
Free PMC article

Abstract

Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan) technology that requires no further manipulations after the PCR step. MethyLight is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10,000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.

Figures

Figure 1
Figure 1
Schematic of the theoretical basis of MethyLight technology. Genomic DNA is first chemically modified by sodium bisulfite. This generates methylation-dependent sequence differences at CpG dinucleotides by converting unmethylated cytosine residues (locations indicated by white circles) to uracil, while methylated cytosine residues (locations indicated by black circles) are retained as cytosine. Fluorescence-based PCR is then performed with primers that either overlap CpG methylation sites or that do not overlap any CpG dinucleotides. Sequence discrimination can occur either at the level of the PCR amplification process or at the level of the probe hybridization process, or both. Sequence discrimination at the PCR amplification level requires the primers and probe (application D), or just the primers (application C), to overlap potential methylation sites (CpG dinucleotides). Only two [fully methylated (M) and fully unmethylated (U)] of the many theoretical methylation permutations are shown. The MethyLight assay can also be designed such that sequence discrimination does not occur at the PCR amplification level. If neither the primers nor the probe overlap sites of CpG dinucleotides (application A), then no methylation-dependent sequence discrimination occurs at the PCR amplification or probe hybridization level. This reaction represents amplification of the converted genomic DNA without bias to methylation status, which can serve as a control for the amount of input DNA. When just the probe overlaps methylation sites (application B), then sequence discrimination can occur through probe hybridization. The design of separate probes for each sequence variant resulting from different methylation patterns (22 = 4 probes in the case of two CpGs, as illustrated) can potentially serve as a quantitative version of the MethyLight technology.
Figure 2
Figure 2
Comparison of the MethyLight assay to a conventional COBRA assay. (A) COBRA gel used to quantitatively determine the level of DNA methylation at the ESR1 locus in DNAs of known methylation status [sperm (unmethylated) and HCT116 (methylated)] The relative amounts of the cleaved products are indicated below the gel. The 56-bp fragment represents DNA molecules in which the TaqI site proximal to the hybridization probe (black box) is methylated in the original genomic DNA. The 86-bp fragment represents DNA molecules in which the proximal TaqI site is unmethylated and the distal site is methylated. (B) A summary of the COBRA results comparing them to the absolute results obtained with the methylated and unmethylated version of the MethyLight assay (application D in Fig. 1). +, a positive absolute value determined by the MethyLight assay; 0, a lack of detectable MethyLight amplification. For the bisulfite-treated samples, the MYOD1 MethyLight assay was performed in parallel to control for the amount of input DNA (application A in Fig. 1). For the untreated samples, the ACTB primers described for the RT–PCR reactions were used as a control to verify the input of unconverted DNA samples. (The ACTB primers do not span an intron.) No PCR indicates that, as expected, no PCR product was obtained on unconverted genomic DNA with COBRA primers designed to amplify bisulfite-converted DNA sequences.
Figure 3
Figure 3
Determination of the specificity of the MethyLight oligonucleotides. Eight different combinations of forward primer, probe and reverse primer were tested on DNA samples with known methylation or lack of methylation at the ESR1 locus. (A) The nomenclature used for the combinations of the ESR1 oligos. U refers to the oligo sequence that anneals with bisulfite-converted unmethylated DNA, while M refers to the methylated version. Position 1 indicates the forward PCR primer, position 2 the probe and position 3 the reverse primer. The combinations used for the eight reactions are shown below each pair of bars, representing duplicate experiments. The MethyLight results are expressed as ratios between the ESR1 values and the MYOD1 control values. (B) An analysis of human sperm DNA. (C) An analysis of DNA obtained from the human colorectal cancer cell line HCT116.
Figure 4
Figure 4
Test of the sensitivity and quantitative accuracy of the MethyLight technique. Human sperm DNA that had been fully methylated by treatment with SssI methyltransferase in vitro was serially diluted in 10-fold increments up to 1:100 000 with untreated, unmethylated human sperm DNA. (A) Subsequent ESR1 MethyLight analysis of these samples, in which the increasing dilutions are indicated by decreasing shades of gray squares, as shown on the right. An ACTB MethyLight control reaction (circles) was included to determine the total amounts of input DNA in each dilution. The shade of gray of the circles corresponds to the dilutions shown for the squares on the right. The relative fluorescence (ΔRn) is plotted as a function of cycle number. The threshold used for the calculation of initial template amounts is indicated by the dark horizontal line (Materials and Methods). (B) ESR1/ACTB ratios obtained for each dilution, plotted against the dilution factor.
Figure 5
Figure 5
Test of the reproducibility of the Methylight reactions. MethyLight assays were performed in eight independent reactions to determine the reproducibility on samples of complex origin. A primary human colorectal adenocarcinoma and matched normal mucosa was used for this purpose (samples 10N and 10T shown in Fig. 6). The results shown in this figure represent the raw values obtained in the MethyLight reaction. The values have been plate-normalized (Materials and Methods), but not corrected for input DNA. The bars indicate the mean values obtained for the eight separate reactions. The error bars represent the standard error of the mean.
Figure 6
Figure 6
Comparison of MLH1 expression, microsatellite instability and MLH1 promoter methylation of 25 matched-paired human colorectal samples. (A) (Upper) MLH1 expression levels measured by quantitative, real time RT–PCR (TaqMan®) in matched normal (hatched bars) and tumor (solid black bars) colorectal samples. The expression levels are displayed as a ratio between MLH1 and ACTB measurements. MSI status is indicated by the circles located between the two charts. A black circle denotes MSI positivity, while an open circle indicates that the sample is MSI negative, as determined by analysis of the BAT25 and BAT26 loci. (Lower) Methylation status of the MLH1 locus as determined by MethyLight assay. The methylation levels are represented as the ratio between the MLH1 methylated reaction and the MYOD1 reaction. (B) Summary of the results of bisulfite genomic sequencing of the 12 CpG sites covered by the three MLH1 MethyLight oligonucleotides for the three tumor samples (12T, 17T and 19T) indicated in (A). Two CpG dinucleotides within the MethyLight amplicon are not covered by any of the three oligos. Eight clones are shown for each sample. Black circles denote methylated cytosines while white circles denote unmethylated cytosines at the indicated CpG dinucleotides. The third CpG in clone 8 of sample 12T could not be conclusively read in that sequence and is indicated by a gray circle.

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