Research Projects

The project proposal aims to develop an advanced single-molecule optical microscopy platform based on smFRET (single-molecule Förster Resonance Energy Transfer) and SMP (single-molecule photobleaching) for DNA fingerprinting. This sensitive technique will provide bias-free, accurate results that would overcome the limitations of traditional PCR-based methods. DNA fingerprinting forms the backbone of modern forensic science and is widely used for applications such as criminal identification and paternity testing. It typically relies on the analysis of Short Tandem Repeats (STRs), which serve as unique genetic markers for individual identification. However, conventional STR analysis relies heavily on enzymatic amplification (e.g., PCR), which is prone to failure when DNA is degraded, chemically modified, or present in trace amounts. These challenges are particularly significant in criminal investigations or post-disaster scenarios, where biological samples are often compromised. This highlights the critical need for robust, amplification-free DNA identification platforms that can directly analyse native DNA at the single-molecule level. Our platform will establish a novel STR analysis method that: 1. Enables amplification-free, direct quantification of STRs at the single-molecule level. 2. Retains high specificity and sensitivity even with degraded or low-concentration DNA. 3. Integrates orthogonal single-molecule readouts for enhanced accuracy and cross-validation. The key experimental modules include: 1. smFRET-Based Distance Profiling–Measuring inter-fluorophore distances that vary with STR length. 2. Single-Molecule Photobleaching–Counting discrete photobleaching steps to validate fluorophore number and STR count. 3. Sensor Design and Assembly–Fabricating DNA nano-sensor constructs optimized for STR detection. 4. iSiMREPS (intramolecular Single Molecule Recognition through Equilibrium Poisson Sampling) Analysis–Using binding kinetics and FRET fluctuation profiles to generate STR-specific kinetic fingerprints. 5. Cross-Validation – Correlating smFRET and SMP results with conventional STR typing (e.g., NGS) to benchmark performance. The platform’s readouts—FRET distances, photobleaching step-counts, and kinetic fingerprints—will collectively offer robust, quantitative STR analysis. This amplification-free strategy will: • Enable direct DNA profiling from degraded forensic or clinical samples by avoiding PCR-induced biases. • Advance the understanding of probe-DNA recognition, binding kinetics, and DNA nanostructure behaviour in real biological samples. • Support biomedical applications, including diagnosis of neurological disorders like Huntington’s disease caused by repeat expansions. By integrating distance, stoichiometry, and dynamics at the single-molecule level, this platform redefines DNA profiling as a high-resolution, amplification-free molecular diagnostic tool with broad applications in forensics, diagnostics, and biosecurity.