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Precise quantitation of DNA methylation by HRM analysis using EZ qPCR/HRM PreMix
Sponsored,vendor-submitted protocol    Sponsored by Zymo Research Corporation    Published in November 2009 (p.27) DOI: 10.2144/000113293

Abstract

High Resolution Melt (HRM) analysis can be used to differentiate DNAs based on variations in primary sequence for applications such as genotyping, SNP analysis, and DNA methylation analysis. Zymo Research has developed the EZ qPCR/HRM PreMix, featuring a robust hot-start polymerase and an intense dsDNA binding dye, for streamlined and precise qPCR and HRM applications. Use of the EZ qPCR/HRM PreMix for measuring DNA methylation of MGMT in two cancer cell lines is described below.

Materials

EZ qPCR/HRM PreMix (Cat. no. E2005; Zymo Research Corporation)

EZ DNA Methylation-Direct™ Kit (Cat. no. D5020-D5023; Zymo Research Corporation)

96-Well Plate (white) w/ Cover Films (Cat. no. 04729692001; Roche Diagnostics, Indianapolis, IN, USA)

Human Methylated/Non-methylated DNA Set (Cat. no. D5014; Zymo Research Corporation)

MGMT Primer Set designed in-house and manufactured by IDT (Integrated DNA Technologies, Coralville, IA, USA)

A549 (lung carcinoma) and HCT 116 (colorectal carcinoma) DNA (ATCC, Manassas, VA, USA)

LightCycler® 480 II (Cat. no. 05015278001; Roche Diagnostics, Indianapolis, IN, USA)

Method

Figure 1. Melting curves of amplicons. Fluorescence vs. temperature was plotted for each data point obtained from the HRM Data Acquisition step following PCR. The raw data indicate that melting temperature varies directly with the amount of methylation in the DNA source.

The following was performed using a LightCycler® 480 II (Roche), although similar results (data not shown) can be obtained using a CFX96™ instrument (Bio-Rad).

1.A549, HCT 116, and methylated DNA standards were bisulfite-converted according to the EZ DNA Methylation-Direct™ Kit protocol. Bisulfite-treated, methylated, and non-methylated DNAs were mixed in equal proportions in the case of the “0%/100% Methylated Mix” sample.

2.Primers were designed with the following parameters: 1) to generate amplicons between 200-400 bp, 2) to comprise 25–35 bp with high G content, and 3) to have Tms over 50°C (ideally between 54–59°C).

3.PCR of Bisulfite-treated DNAs (Table 1).

4.The qPCR/HRM protocol entailed a 10-minute denaturation at 95°C, followed by 45 cycles of the following 3 steps: 30 s denaturation at 95°C, 1 min annealing at 56°C, and 1 min. extension at 72°C. After a 2-min extension step at 72°C and cooling at 40°C for 30 s, HRM data was acquired while ramping from 65°C to 95°C at a rate of 0.01°C/s.

Results

Table 1: PCR Reagents

Data acquired during the melting of PCR products generated from bisulfite-treated DNAs are plotted in Figure 1. These HRM data were exported using the Roche LightCycler software into a Microsoft Excel spreadsheet, and then normalized (Figure 2) using an algorithm that we designed to highlight the difference in melt profiles between the 0% to 100% methylated DNA samples.

Based on the differences in melt profiles and using the non-methylated and methylated DNAs for reference, normalization of the data enabled quantification of the extent of methylation (percentage) for each sample at the MGMT locus. At this locus, HCT 116 DNA was determined to be 33.7% methylated and the A549 DNA was 83.8% methylated.

Conclusion

Figure 2. Normalized fluorescence of amplicons relative to the non-methylated control. Plot of the data after it was normalized using an algorithm developed at Zymo. The differences in melt profiles are easily discernable and can be used to estimate the percentage of methylation.

Our results illustrate the utility of the EZ qPCR/HRM PreMix in performing HRM for DNA methylation analysis. Critical to the precision and success of this system is the ≥99.5% conversion efficiency and robust amplification of bisulfite-treated DNA afforded by specially designed bisulfite treatment and amplification technologies, respectively. The product and method described here are appropriate for basic research, clinical applications, and high-throughput analysis. For example, we also performed site-specific methylation analysis for multiple loci for several tissues (i.e. breast, colon, liver, and lung). The EZ qPCR/HRM PreMix can also be used for conventional qPCR on any instrument that does not require passive reference dye (Roche LightCycler®, Bio-Rad CFX™, LightScanner®, Rotor-Gene™, etc.).

Zymo Research sells the premix individually (E2005, E2006) or as part of the EZ DNA Methylation-Direct™ qPCR/HRM Kit (D5300). The latter comprises a workflow that enables the researcher to start directly from blood or tissue samples and end in DNA methylation analysis/quantitation via HRM in just one day.

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