Discrimination of base differences in oligonucleotides using mid-infrared spectroscopy and multivariate analysis.
Attenuated total reflection Fourier transform-infrared (ATR-FTIR) spectroscopy was employed to interrogate a panel of simple oligonucleotides designed to contain various base differences; combined with subsequent multivariate analysis, we set out to determine whether the specificity of this approach would point to a novel means for mutation detection. Oligonucleotides were designed that were 15 bases in length and contained various combinations of purines (adenine, guanine) or pyrimidines (cytosine, thymine). These were applied to 1 cm x 1 cm low-E reflective glass slides, and triplicate samples were interrogated using ATR-FTIR spectroscopy. Per oligonucleotide sample, 10 independent spectral acquisitions were obtained. Prior to multivariate analysis, infrared spectra were baseline-corrected and vector normalized over the 1750-760 cm(-1) region specific to the chemical bonds of organic molecules. Spectral categories were then analyzed using principal component analysis (PCA) followed by linear discriminant analysis (LDA). Scores plots revealed that PCA-LDA clearly segregated different oligonucleotide sequences, even in the presence of a single base difference. Loadings plots confirmed the chemical entities associated with distinguishing base differences. These results suggest that mid-IR spectroscopy might have future roles in interrogating polymorphic forms of a DNA template.
Kelly
Jemma G
JG
Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, UK.
Martin-Hirsch
Pierre L
PL
Martin
Francis L
FL
D001483:Base Sequence; D016002:Discriminant Analysis; D015999:Multivariate Analysis; D009841:Oligonucleotides / Q000737:chemistry*; D025341:Principal Component Analysis; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D009841:Oligonucleotides