DNA Methylation 101

DNA Methylation is a phrase that has gained in popularity lately, but if you’re like me, maybe you’re wondering what DNA methylation is, and more importantly, how can it be used for forensic applications? If that sounds familiar, than this post is for you, because we’re going to tackle those very questions.



Written by: Tara Luther, Promega



Forensic DNA typing has proven to be an invaluable tool in solving crime and finding justice for victims, but it has its limitations. For instance, what if there is no suspect match in a database, or what if we need to know exactly what type of biological fluid has been found at a crime scene? Additionally, recent news stories have shown that when dealing with suspects who have an identical twin, it’s very difficult (and expensive) to determine who was the culprit, and even if you can, will it be accepted in court?

While DNA methylation cannot overcome all of these obstacles, it can provide additional information to help identify a suspect and then bring them to justice. Read on to discover what DNA methylation is and how it can be used in addition to traditional DNA typing.


What is DNA Methylation?

To understand DNA methylation, you first have to know a bit about epigenetics. Epigenetics is a broad term used to describe reversible modifications to the genome. In addition to each individuals’ genetic blueprint, the epigenetic code is inherited patterns that include an additional layer of information. A widely accepted definition is the “study of processes that produce a heritable phenotype that does not strictly depend on the DNA sequence”.

Epigenetic patterns are preserved during cell division, but they can change over an individual’s lifetime. These changes can be due to age, environmental factors, and an individual’s health. Remember this as we discuss benefits to DNA methylation later.

Now to the topic at hand. In the human genome, DNA methylation is a necessary biochemical process for normal development. Without DNA methylation, normal cells would stop growing or die. It involves adding a methyl group at the 5’ position of cytosine residues in CpG dinucleotides. The methylation process helps in the stabilization of the condensed chromatin, and has also been found to regulate gene expression. Un-methylated DNA sequences are treated with sodium bisulfite resulting in two unique DNA sequences – one methylated, and one un-methylated. Conversion of DNA using sodium bisulfite allows for the qualitative and quantitative analysis of individual CpG sites.


How Can DNA Methylation be applied to Forensic Casework?

Discrimination of Bodily Fluid

There are multiple types of body fluid that can be found at a crime scene: blood, saliva, semen, vaginal fluids, sweat, or urine. While traditional DNA typing can be used to match a suspect to a crime scene, sometimes it is also necessary to determine the type of bodily fluid found as a means of reconstructing the crime scene. While this can be done via traditional DNA typing, the part of the sample used for the test would be consumed. This can be a problem when there was only a small sample to begin with.

This is where DNA methylation can play an important role. It is highly cell-specific, meaning methylation patterns found in the same tissue type are more consistent among different individuals than it is among different tissues within the same person. By looking at the patterns of methylation, analysts can determine the type of fluid present without destroying the sample.

In the video below, Joana Antunes of Florida International University describes her work with DNA methylation and bodily fluid identification:


Ancestral Information

In their article, Genomic DNA Methylation among Women in a Multiethnic New York City Birth Cohert, Terry et al determined that Black individuals displayed lower levels of methylation than White or Hispanic individuals. This information can be useful when creating a phenotypic profile for a potential suspect, unidentified remain, or in the case of missing persons. It can also be used as an additional tool in the re-examination of exhausted cold cases.


DNA methylation may be able to provide information when DNA typing can't. Share on X


Determination of Age

DNA methylation levels change throughout an individual’s lifetime. While levels increase during an individual’s first year of life and remain steady throughout adolescence, they gradually decrease across the genome after adulthood is reached. Based on this knowledge, analysts are able to predict the age of an individual within 5 years.

In addition, DNA methylation can also be used to determine the rate of aging for an individual. Deviations from standard levels could indicate underlying health or physical conditions that can’t be determined from autopsy alone.

For more information, read this blog post.


Identification of Twins

Individualizing identical twins has been a difficulty for analysts since they contain the same DNA. Since DNA methylation is impacted by environmental and health factors, the twin’s methylation patterns will differ. Looking at those patterns make it possible to distinguish identical twins.


Determining Sex

Naito et al describe a study they performed in their article, Sex Determination Using the Hypomethylation of a Human Macro-Satellite DXZ4 in Female Cells, which has forensic applications in determining if a sample was from a female. The study analyzed methylation patterns of DXZ4, an X chromosome-specific region. The sequence showed low degrees of methylation on the inactive X, but hypermethylation on the active X chromosome. This protocol was also quite sensitive, so only a minute amount of DNA was deemed necessary for accurate sex typing.


While there are many promising applications for DNA methylation in forensics, some challenges still remain. For instance, while this may be a great tool to narrow down a pool of suspects for investigators, the sensitivity of the marker and detection methods are among issues to consider before DNA methylation can be used in the courtroom. Consideration must also be made when mixed samples are presented, and all markers used must show high sensitivity and specificity, as forensic samples are often small or degraded. As with all new technologies, however, perhaps one day DNA methylation will become commonplace in the lab.