Quantum Method for Fluorescence Background Removal in DNA Melting Analysis

Abstract

Fluorescent high-resolution DNA melting analysis is a robust method of genotyping and mutation scanning. However, removing background fluorescence is important for accurate classification and to correctly display helicity. Linear baseline extrapolation, commonly used with absorbance, often fails at low temperatures when fluorescence is used. A new quantum method of background removal based on the inherent decrease of fluorescence with temperature is described. Absorbance and fluorescence melting curves were compared using synthetic targets including hairpins, unlabeled probes, and a 50 bp duplex. In addition, the quantum method was compared to a previously described exponential method for analysis of genotyping data produced after polymerase chain reaction (PCR), including those from small amplicons, unlabeled probes, and snapback primers. The quantum method best matched absorbance data and predicted helicity, with the exponential method displaying low-temperature bulges and domain artifacts that can lead to incorrect genotyping. When two melting domains were widely separated, quantum analysis produced a flat baseline between domains, while exponential analysis was temperature-dependent. Both methods have little effect on the melting temperature (Tm) although some differences were significant (hairpin Tm values increased 0.7 °C by the quantum method and decreased 1.5 °C by exponential method, p = 0.01). However, peak heights on derivative plots were strongly algorithm-dependent, with exponential analysis enhancing low-temperature peaks while dampening high-temperature peaks. Quantum-analyzed fluorescence curves were a better match to absorbance data in terms of shape, area, and peak height compared to other methods, indicating that DNA helicity is best approximated by the quantum method.