DNA methylation assays
Epigenetics is a study of the heritable gene expression changes that do not involve alternations in DNA sequence. Prefix epi- signifies “on top of” or “in addition to”, hence epi-genetic is understood as something on top of genetics that changes the expression of the genes.
- 1) DNA methylation
- 2) Treatment with bisulfite modification
- 3) DNA melting temperature
- 4) How to analyze the results from High Resolution Melting
- 5) Controls in methylation detection experiments
1) DNA methylation
Enzymatic addition of methyl groups (CH3-) to the DNA molecule is referred to as DNA methylation. In mammals the methyl groups are almost exclusively added to cytosines at CpG dinucleotides. Specific genomic regions have a high density of CpG dinucleotides and are defined as CpG islands (CGI). When CGIs are located in promoter regions DNA methylation typically acts to repress gene transcription.
Abnormal changes in DNA methylation have been identified in many different diseases as cancer, inflammatory, autoimmune, psychiatric, cardiovascular, and age-related diseases.
2) Treatment with bisulfite modification
During DNA replication (cell division) methyl groups (DNA methylation) are resynthesized on the newly replicated strand by one of the enzymes called DNA Methyl Transferases (DNMT). Those enzymes are not present in standard PCR reaction. Therefore, a PCR product obtained from amplification of a specific locus does not contain information about methylation status of the cytosines and the methylation information is lost. To analyze the methylation pattern of cytosines within the locus of interest the information about which cytosines are methylated needs to be preserved before PCR amplification is performed. Sodium bisulfite deaminates non-methylated cytosines to uracil and leaves methylated cytosines untouched (in other words methylated cytosines are resistant to modification induced by sodium bisulfite).
3) DNA melting temperature
Dissociation of the double stranded DNA helix into single coils is referred to as DNA melting. It can be accomplished by simply heating double stranded DNA. The temperature at which the DNA strands dissociates into single coils depends on the number of hydrogen bonds holding the complementary strands. There are two hydrogen bonds between adenine (A) and thymine (T) and three bonds between guanine (G) and cytosine (C). Therefore, in principle the more G and C in the sequence the higher temperature is needed to melt a given DNA fragment.
How to detect difference in melting temperature of PCR products?
The most commonly used method to determine the melting temperature of a PCR product is to subject the product to a temperature gradient in the presence of intercalating dye. The intercalating dyes are chemicals that only emit light when bound to double stranded DNA. In a typical melting experiment, a PCR product is mixed with an intercalating dye, and fluorescence emitted by this mix is monitored as the sample is slowly heated (subjected to a temperature gradient).
The outcome of the analysis is a curve displaying fluorescence changes emitted by the sample over the range of temperature that the sample was subjected to, commonly referred to as a melting profile. At the beginning of the melting experiment the temperature is low and all PCR product in the sample is double stranded. Thus, we observe high levels of florescence from the sample. We continue to observe high levels of fluorescence, as the temperature increases up to the point, where all hydrogen bonds within the PCR fragment are broken and the amount of double stranded PCR product drastically decreases. Consequently, we observe a sharp decrease in the detected fluorescence level. At a high temperature there is no double stranded PCR product in the sample and the fluorescence levels are close to 0. The temperature at which we observe the sharp drop in the fluorescence depends on the number of hydrogen bands in the analyzed PCR product and hence is specific to analyzed fragment.
4) How to analyze the results of High Resolution Melting
The number of cytosines (C) in the PCR product amplified from a bisulfite modified template depends on methylation status of cytosines within that template before bisulfite modification. In principle, after PCR amplification a C rich PCR product is obtained if the amplified locus was methylated and a T rich PCR product if the locus was not methylated. These PCR products will display different melting profiles (see also: What is DNA melting?) when subjected to MS-HRM analyses. The PCR product amplified form non-methylated version of specific locus will have relatively low melting temperature and melt earlier in the temperature gradient than the PCR product amplified from the methylated version of the same locus which will melt at relatively higher temperature.
5) Controls in methylation detection experiments
Following good laboratory practice any experiment needs to be properly controlled. In PCR based methylation screening experiments two controls are necessary: methylated (positive) and non-methylated (negative). Those controls are normally chemically modified DNA templates consisting of methylated and non-methylated versions of the locus of interest and are reference points for the analyses of an unknown sample.
In the Methylation-Sensitive High-Resolution Melting (MS-HRM) protocol methylation status of a screened sample is assessed by comparison of the melting profile (see also: What is DNA melting?) of the PCR product amplified from a screened sample (Figure 1 – green) with the melting profiles of PCR products amplified from methylated (Figure 1 – blue) and non-methylated (Figure 1 – red) controls.