Agathaw Christie - DNA-typing with High-Resolution Melting Analysis - Methylation and STRs

Abstract

In forensics, identifying and comparing DNA is common practice. One way to analyse DNA is through high-resolution DNA melting. When melting DNA experimentally, the rate of a given sequence of DNA denaturing from its double-helix form into two separate strands is measured as a function of temperature.

In this thesis, the application of high-resolution DNA melting analysis to distinguish individuals through their DNA in forensics is investigated by simulating the melting behaviour with the Poland-Scheraga model. Previously this has been done assuming the ends of the DNA strands are clamped. We assume free ends. We will be looking at DNA sequences of 80 - 300 base pairs.

The differentiability of DNA sequences is examined based on number of short tandem repeats (STRs) or degree of methylation. STRs are blocks of DNA bases that repeat to varying amounts depending on the individual. Methylation is a lifestyle-affected stochastic process where a phosphate attaches itself to certain sites in the DNA.

We here introduce the $Q$-value which is used to create a fingerprint of an individual. It is calculated by multiplying the temperature derivative of the melting curves by the weighting function and summing across the temperature. The $Q$-value is a scalar based on the melting behaviour at temperatures in the melting process with the highest difference across the range of melting curves. Going through the number of STRs or degrees of methylation, a range of melting curves of each sequence is simulated. Based on the temperature derivatives of these melting curves, a weighting function is created which plots the variance of the melting curves as a function of temperature.

Due to the maternal and paternal part of DNA, each sequence is represented twice. For each sequence, the two sequences can be the same (homozygous) or different (heterozygous). In homozygous DNA, we find that the $Q$-value changes monotonously with increasing number of STRs and can distinguish between DNA sequences. In heterozygous DNA, we find that the $Q$-value increases with increasing number of summed STRs of both alleles. The $Q$-method is able to distinguish between sequences where the difference between the number of summed STRs is greater than 3.

In the case of methylation typing, we show that the $Q$-method is able to distinguish between sequences where the difference between the degree of methylation is greater than 32$\%$.

Looking forward, the melting curve of a known sequence needs to be experimentally obtained and compared against the model-based simulation.

Popular Abstract

Agathaw Christie - DNA melting in forensics

Old man Hercule was found murdered in his lake house. You have been chosen to find the culprit. You have found DNA at the crime scene and have three suspects: the gardener and a set of twins that are distant relatives of Hercule.
DNA is made up of four different bases: adenine (A), thymine (T), cytosine (C), and guanine (G). It is structured in a double helix shape: two long strands of bases winding around one another. Bases on opposing strands bond together forming base pairs which maintains the structure. In many places in DNA, there are short blocks of bases that repeat called short-tandem repeats (STR). In a sequence called D13S317, the block TATC repeats 5-16 times. The number of STRs varies between individuals. By looking at around 20 such sequences and determining the amount of STRs in each, a person can be identified.
The number of STRs is identical between twins. Over time, phosphate molecules randomly attach to certain bases. This process is affected by your lifestyle and is called methylation. Thus, one way to distinguish twins is by looking at the methylation of the DNA.
At the moment, the common way of distinguishing DNA is electrophoresis. The strands are placed in a gel and an electric field is applied which makes them move. The longer the strand, the less distance it travels through the gel. By measuring the distance the strand travelled, its length and the amount of STRs can be determined. While this method works reliably, it is slow, costly, and can’t differentiate twins
We will distinguish the number of STRs and the degree of methylation through a process called “High Resolution DNA Melting Analysis”. A molecule is attached to each base pair which glows when the bases are connected. The temperature is increased from 50°C to 110°C, causing the base pairs to detach or “melt”. By measuring the light emission, we can see at what temperature and how fast the strands detach from one another. As STRs have a lower melting temperature than the rest of the DNA sequence, varying amounts of STRs will change the melting behaviour. Due to a certain treatment, methylated base pairs melt later, allowing to differentiate degrees of methylation.
In this thesis, the melting of several sequences was simulated with a MATLAB code using a model that takes into account how the base pairs connect, how neighbouring bases in a strand interact, and other strand behaviours when the temperature is increased. First, we plot how fast the sequence melts for different amounts of STRs. The sequence D13S317 from earlier was plotted for TATC repeating 5-16 times. From this, a weighting curve is created that tells you at which temperature the difference in melting behaviour is the biggest. Each sequence will have a unique weighting curve. By multiplying the melting behaviour with the weighting curve and taking the sum along the temperatures, we get a number: the Q-value. The same method is used for methylation, associating Q-values with different degrees of methylation.
You reveal that the murderer must be one of the twins! You multiply the crime scene DNA’s melting behaviour with the sequence’s weighting curve and get a Q-value for each sequence. You compare the set of Q-values from the crime scene with the Q-values from your suspects. You are now quite certain the murderer is the twin Achille Poirot.

• author: Eckle, Lauri
• supervisor: Tobias Ambjörnsson
• organization: Department of Physics
• course: FYSK04 20251
• year: 2025
• type: M2 - Bachelor Degree
• subject: Physics and Astronomy; Biology and Life Sciences
• keywords: DNA melting, HRMA, Poland-Sheraga, methylation, STR, High Resolution Melting
• language: English
• id: 9209295
• date added to LUP: 2025-08-06 08:59:49
• date last changed: 2025-08-06 08:59:49