DNA methylation, the addition of a methyl group to the 5′ position of cytosine in CpG dinucleotides, is essential for normal development and function of mammalian cells. Alterations in DNA methylation have been implicated in numerous diseases including cancer, where aberrant gene promoter hypermethylation is associated with transcriptional silencing of genes (1). It is an early event in neoplastic progression found in virtually every type of human cancer and can be detected in biological fluids prior to symptomatic disease (2,3). Identifying cancer in its earliest stages could greatly reduce morbidity and mortality. Biomarkers based on gene promoter hypermethylation currently are being developed and evaluated as molecular markers for cancer detection and therapeutic efficacy. Methylation-specific PCR (MSP) is the most widely used technique to study CpG island methylation. It is a sensitive and specific assay relying on bisulfite modification (BSM) to differentiate methylated and unmethylated CpGs. BSM converts unmethylated cytosines in genomic DNA to uracils, while methylated cytosines remain unchanged. MSP typically can detect one methylated allele in 1000 unmethylated alleles (4). We previously developed a nested MSP method that increased MSP sensitivity approximately 50-fold (5). First-stage PCR primers in nested MSP recognize a bisulfite-modified template but do not discriminate between methylated and unmethylated alleles. Multiple genes can be amplified in a first-stage nested MSP (6,7). In the second stage, two PCRs are conducted. One uses primers specific for an unmethylated template, and the other uses primers specific for a methylated template. Agarose gel electrophoresis is used to separate and visualize the PCR products. The presence of a stage 1 product or a stage 2 unmethylated product verifies target sequence amplification. We have used this approach to detect aberrant tumor-specific gene promoter hyper-methylation in sputum DNA from patients prior to clinical diagnoses of lung cancer (5). However, currently only the stage 1 PCR of nested MSP is multiplexed, separate stage 2 MSPs are required to determine the methylation status for an individual gene. This approach is both time-consuming and costly, thus limiting the efficiency of conducting population-based screening studies. We have addressed these limitations by developing a nested MSP method that simultaneously assays the methylation status of multiple gene promoters and combined it with nongel-based detection of the PCR products. The nongel-based analysis relies on denaturing high-performance liquid chromatography (DHPLC) using the WAVE® instrument (Transgenomic, Omaha, NE, USA). Method set up is straightforward, semi-automated, allows simultaneous examination of the promoter methylation status for multiple genes, uses the same DNA quantity as a nested single-gene MSP analysis, and can utilize formalin-fixed paraffin-embedded DNA from multiple tissue sources.

A description of the nested multigene MSP/DHPLC method is provided, and the results are compared with those obtained with the nested single-gene MSP/agarose gel analysis method. The gene promoter hypermethylation status of p16, RASSF1A, and DAPK was analyzed in 40 sputum samples from current and former smokers at risk for lung cancer and in 20 formalin-fixed paraffin-embedded breast tumor samples. Each of the genes is a tumor suppressor affecting different cellular pathways: (i) p16, cell cycle control; (ii) DAPK, cell adherence and metastasis processes; and (iii) RASSF1A, apoptosis (8). All have important roles in carcinogenesis and are silenced by aberrant promoter methylation (8,9). To demonstrate the utility of this method, the samples chosen were predominately methylated, and thus, the methylation prevalence is not indicative of that normally detected for these genes in either tissue. Primer sequences for the nested three-gene MSP method are listed in (Table 1). Stage 1 and stage 2 unmethylated PCR conditions consisted of a 10-min 94°C denaturation step, followed by 40 cycles of a 30-s 94°C denaturation step, a 30-s 60°C annealing step, a 30-s 72°C extension step, and a final elongation step of 72°C for 10 min. In the stage 2 methylated PCR, times for the second denaturation step, the annealing step, and the extension step were decreased to 15 s. All PCRs were conducted in a final volume of 50 µL. The first stage PCR used approximately 150 ng bisulfite-modified DNA, the second stage unmethylated PCR used 1 µL of a 1:50 dilution of stage 1 PCR product, and the second stage methylated PCR used 5 µL of a 1:50 dilution of stage 1 PCR product as a template. Differences in reaction conditions for stage 2 methylated PCR between the nested single-gene MSP and the nested three-gene MSP method involve alterations in primer, Taq DNA polymerase, and MgCl₂ concentrations. Specifically, p16 and DAPK primer concentrations were reduced by 50% to 10 µM, the amount of AmpliTaq Gold® DNA Polymerase (Applied Biosystems, Foster City, CA, USA) was doubled to 5 U, and an additional 7.5 nmol of MgCl₂ were added.