RESUMEN
Methylation status plays important roles in the regulation of gene expression and significantly influences the dynamics, bending and flexibility of DNA. The aim of this study was to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) or Raman spectroscopy with subsequent multivariate analysis could determine methylation patterning in oligonucleotides variously containing 5-methylcytosine, cytosine and guanine bases. Applied to Low-E reflective glass slides, 10 independent spectral acquisitions were acquired per oligonucleotide sample. Resultant spectra were baseline-corrected and vector normalised over the 1750 cm(-1) -760 cm(-1) (for ATR-FTIR spectroscopy) or the 1750 cm(-1) -600 cm(-1) (for Raman spectroscopy) regions. Data were then analysed using principal component analysis (PCA) coupled with linear discriminant analysis (LDA). Exploiting this approach, biomolecular signatures enabling sensitive and specific discrimination of methylation patterning were derived. For DNA sequence and methylation analysis, this approach has the potential to be an important tool, especially when material is scarce.
Asunto(s)
Metilación de ADN , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Espectrometría Raman/métodos , Secuencia de Bases , ADN de Cadena Simple/genética , Análisis Discriminante , Análisis Multivariante , Análisis de Componente PrincipalRESUMEN
The Syrian hamster embryo (SHE) assay (pH 6.7) is an in vitro candidate to replace in vivo carcinogenicity tests. However, the conventional method of visual scoring of foci (non-transformed vs. transformed colonies) can be time-consuming and is open to subjectivity. Infrared (IR) spectroscopy has the potential to provide objective assessment of such SHE colonies with the added advantage of potentially providing mechanistic information. In this study, SHE cells were treated with one of eight different chemical regimens, allowed in culture to attach and form foci on IR-reflective glass slides; these were subsequently interrogated by attenuated total reflection (ATR) Fourier-transform IR (FTIR) spectroscopy. Derived mid-IR spectra (n = 13,406) were subjected to chemometric analysis focusing primarily on the extraction of biochemical information related to test agent treatment and/or morphological transformation. The use of ATR-FTIR spectroscopy with chemometrics to analyze the SHE assay is a novel approach to toxicological assessment.