Key factors in FTIR spectroscopic analysis of DNA: the sampling technique, pretreatment temperature and sample concentration

Abstract

Fourier transform infrared (FTIR) spectroscopy has been considered as a powerful tool for analysing the characteristics of deoxyribonucleic acid (DNA) regardless of physical states, sample amounts and the molecular weight of DNA. However, FTIR spectroscopic analysis of DNA might be influenced by the stability and conformational integrity of deoxyribonucleic acid. This work systematically investigated the key factors in FTIR spectroscopic analysis of DNA and explored the influence of FTIR acquisition parameters, including FTIR sampling techniques (traditional KBr-FTIR, high throughput (HT)-FTIR, Micro-FTIR-point, and Micro-FTIR-image), pretreatment temperature, and sample concentration, on calf thymus DNA (CT-DNA). The results showed that the FTIR sampling techniques had a significant influence on the spectral characteristics, spectral quality, and sampling efficiency. Furthermore, HT-FTIR technique enabled rapid automated collection of spectra and was selected for obtaining better spectral accuracy and repeatability. Additionally, a pretreatment temperature of 30 °C was selected to dry DNA, because the continuous increase of temperature had a great impact on the spectral analysis of DNA. The sample concentration was also proven to have a great effect on the spectral repeatability and SNR, especially for the initial sample amounts, where optimum sample amounts (100 μg) were selected. These fundamental studies provide proof of a spectral collection that further supports subtle DNA structural analysis by FTIR spectroscopy.