2Neurosciences Research Group, School of Medicine, Universidad Nacional de Colombia, Bogotá, Colombia
3Biomedical Sciences Doctoral Program, School of Medicine, Universidad Nacional de Colombia, Bogotá, Colombia
4Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada
Comprehensive analysis of DNA methylation patterns is critical for understanding the molecular basis of many human diseases. While hundreds of PCR-based DNA methylation studies are published every year, the selection and implementation of appropriate methods for these studies can be challenging for molecular genetics researchers not yet familiar with methylation analysis. Here we review the most commonly used PCR-based DNA methylation analysis techniques: bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), MethyLight, and methylation-sensitive high resolution melting (MS-HRM). We provide critical analysis of the strengths and weaknesses of each approach as well as a series of guidelines to assist in selecting and implementing an appropriate method.
DNA methylation is the most extensively studied mechanism for epigenetic gene regulation (1). Recent studies have shown that DNA methylation plays an important role in a number of physiological processes as well as common diseases such as cancer and neurodegenerative disorders (2,3). In mammals, DNA methylation occurs at the C-5 position of cytosine in CpG dinucleotide sequences (Figure 1) (1), which are mainly concentrated in regions known as CpG islands. Methylation in CpG islands within gene promoters usually leads to gene silencing. More recently, DNA methylation in regions located up to 2 kb from known CpG islands (called CpG island shores) has also shown a strong correlation with gene expression (4).
At present, the vast array of platforms available to study DNA methylation present a challenge for scientists who wish to enter this field (5). Among the methods for studying DNA methylation in candidate regions, PCR-based approaches have several advantages (6). Here we provide a practical overview of experimental design and analysis for the most common PCR-based DNA methylation techniques: bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), MethyLight, and methylation-sensitive high resolution melting (MS-HRM). These techniques do not need expensive specialized equipment and could be implemented in a typical molecular genetics laboratory.Bisulfite conversion
The first step in almost all protocols for studying DNA methylation is bisulfite conversion of the DNA sequence of interest. Bisulfite conversion occurs through a number of chemical reactions (e.g., sulfonation, deamination, and desulfonation) on the DNA that transform non-methylated cytosines into uracils. Methylated cytosines remain unconverted (Figure 1). Classical DNA conversion protocols are time-consuming, often requiring more than 16 h to complete (7), and require multiple tube changing steps that increase the risk of contamination and human error. Classical protocols also risk losing more than 75% of the starting DNA (8,9) during purification and through single-strand breaks that occur during long incubation steps (7,9).
Commercially available bisulfite conversion kits improve recovery of the converted DNA by using shorter incubation steps and alternative purification procedures (9). These kits also facilitate efficient implementation of the conversion reaction, thereby improving downstream results with PCR-based techniques. Thus, kits are highly recommended, especially for those unfamiliar with this field of study. There are many considerations for selecting a kit, including cost, yield, efficiency, and time. A comparison of the main features of available DNA conversion and methylation control kits is included in Tables 1 and 2.Table 1.
Controls for DNA bisulfite conversion
Evaluation of the quality of converted DNA is recommended when beginning a DNA methylation study; this step is especially important for quantitative PCR-based methods such as MethyLight and MS-HRM. Since bisulfite-treatment can result in DNA fragmentation, thus reducing the number of molecules available for PCR amplification, it is best to test the bisulfite-converted DNA with primer sets that amplify a range of differently sized products. From these products, the ideal amplicon length for downstream analysis can be determined (10), providing information that will aid in primer design.
Incomplete bisulfite conversion will adversely affect the reliability and accuracy of DNA methylation measurements by PCR-based methods (11,12). Therefore, it is necessary to evaluate the efficiency of conversion using commercially available primer sets to amplify the converted DNA (e.g., DAPK1 Catalog #D5014–2, Zymo Research, Orange, CA) (Table 1). The resulting DNA product can be sequenced to verify the efficiency of conversion for all non-CpG cytosines. Alternatively, converted DNA may be amplified with primers designed for the non-converted DNA sequence. In this case, the absence of a PCR amplicon suggests a complete conversion reaction.
Converted DNA must also be quantified prior to downstream PCR applications. The amount of DNA may be determined by spectrophotometric measurements using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA) (13) with settings for single-stranded DNA, or agarose gel electrophoresis and classical UV spectrometric analyses (5). Other more specific methods, such as qPCR (including MethyLight control assays) or PicoGreen may be more reliable and better suited for measuring limited amounts of DNA (14).