DNA Methylation, Melting DNA, and Twins: Advancing Forensic Investigations

DNA profiling, also known as genetic fingerprinting has been crucial to the forensic investigation of crime scenes since the 1980s. Although important to the success of many cases, this technique comes with some limitations, particularly when investigators are trying to match DNA evidence to a twin. Using this method, it is difficult to conclusively determine which twin may be innocent and which may be guilty because of their identical DNA sequence. Scientists at the University of Huddersfield, however, conducted research assessing a useful technique that can differentiate between identical DNA of twins, focusing on DNA methylation and the melting temperature of DNA. Their results were recently published in Analytical Biochemistry.

With the traditional DNA profiling method, the chance that your DNA will match the DNA of someone unrelated to you is around one in a billion. The probability is reduced to 1 in 10,000 for two full siblings, but identical twins have a matching DNA sequence. This may complicate forensic investigation when trying to use DNA to determine which twin actually committed a crime. Dr. Graham Williams and his Forensic Genetics Research Group at the University of Huddersfield, however, have proposed a technique that could solve this issue and help advance crime scene investigation techniques.

As discussed in the blog article by Fides Lay, Ph.D., DNA Methylation Solves Crimes, a DNA methylation-based assay could be used in forensic applications for age prediction, authentication of DNA samples, body fluid identification, and perhaps even differentiating between monozygotic twins. This team of researchers from the University of Huddersfield applied this idea and proposed using a technique to distinguish between the DNA of identical twins based on DNA methylation status and the melting of hydrogen bonds.

Previous techniques such as “mutation analysis” focus on genome-wide sequencing of DNA from both twins, identifying the mutations that may have occurred to the DNA of one twin and not the other. According to Dr. Williams, a mutation, if identified in one twin, could match a specific mutation from a DNA sample taken from the scene of the crime. This is accomplished via bisulfite treatment followed by pyrosequencing. However, this bisulfite sequencing method is very costly for forensic investigators and takes a lot of time to complete. Dr. Williams explains it is unlikely that police forces on a typically tight budget would opt for such an expensive procedure.

This new research focuses on DNA methylation and the degree of difference between twins, which increases over time as they experience different environments. Various exposures to drugs or different work environments, for example, could change the methylation states of their DNA. As we know, DNA methylation is central to the field of epigenetics and can control whether a gene is turned on or off. Honing in on this epigenetic mark, these researchers utilized an inexpensive method to distinguish between the DNA of two identical twins, specifically, DNA methylation-specific high-resolution melt curve analysis.

Dr. Williams explains the procedure: “What HRMA does is to subject the DNA to increasingly high temperatures until the hydrogen bonds break, known as the melting temperature.  The more hydrogen bonds that are present in the DNA, the higher the temperature required to melt them.”

“Consequently, if one DNA sequence is more methylated than the other, then the melting temperatures of the two samples will differ – a difference that can be measured, and which will establish the difference between two identical twins.”

In their study, the research team subjected buccal swabs, or cheek cells, from five sets of monozygotic twins to bisulfite treatment, polymerase chain reaction amplification and high-resolution melting curve analysis that targeted two markers – Alu-E2F3 and Alu-SP. Their results indicated a significant difference between all monozygotic twins for the Alu-E2F3 marker and in four of five monozygotic twins targeting Alu-SP.

Although their research was successful, DNA methylation-specific HRMA comes with some limitations that should be acknowledged. Young twins raised in similar environments would not see much difference in their DNA methylation profiles. In addition, the technique also relies on high sample quantity that may be hard to come by at a crime scene.

Nevertheless, Dr. Williams indicates they have made “substantial progress towards a relatively cheap and quick test for differentiating between identical twins in forensic case work.” In light of this research, investigators could use DNA methylation and the melting temperature of DNA to piece together the clues and be more confident in connecting the guilty twin to the crime.

Source:  Stewart, L., Evans, N., Bexon, K.J., van der Meer, D.J., Williams, G.A. (2015). Differentiating between monozygotic twins through DNA methylation-specific high-resolution melt curve analysis. Analytical Biochemistry, 476: 36.

References: The University of Huddersfield. Crime scene discovery – separating the DNA of identical twins. 20 Apr 2015.