The development of colorimetric assays to determine the identity and frequency of specific nucleobases in DNA oligomers

Abstract

OF DISSERTATION THE DEVELOPMENT OF COLORIMETRIC ASSAYS TO DETERMINE THE IDENTITY AND FREQUENCY OF SPECIFIC NUCLEOBASES IN DNA OLIGOMERS Colorimetric methods combined with color-changing chemical probes are widely used as simple yet effective tools for identifying and quantifying a wide variety of molecules in solution. For nucleic acids (DNA and RNA), perhaps the most commonly used colorimetric probe is potassium permanganate, which can be used to identify single-stranded pyrimidines (thymine and cytosine) in polymers. Unfortunately, permanganate is not an effective probe for identifying purines (adenine and guanine), especially in the presence of the more reactive pyrimidines. Therefore, robust methods for discriminating between the purines remain elusive, thereby creating a barrier toward developing more complex colorimetric applications. In this dissertation, we demonstrate that chromophores such as permanganate and bicinchoninic acid (BCA) and copper, however, when combined with nucleobase-specific chemical cleavage reactions, can be a colorimetric probe for the identification and quantification of cytosines, adenines and/or guanines in single-stranded DNA oligomers, even in the presence of thymines. Furthermore, the reactions are stoichiometric, which allows for the quantification of cytosine, adenine and/or guanine frequency in these oligomers. The BCA/copper reagent detects the reducing sugar, 2-deoxyribose, resulting from the chemical cleavage of a given nucleotide’s N-glycosidic bond. Therefore, these colorimetric assays are effectively detecting abasic sites in DNA oligomers, which are known to occur in damaged DNA. Our analytic approach termed colorimetric identification of exposed nucleic acids (CIENA) combines the use of BCA/copper, permanganate, and diphenylamine chromophores along with digital image capture to identify and quantify each nucleobase within DNA. The digital image color properties are quantified in terms of the image’s hue, saturation, and lightness using the CIELAB color space and ∆E quantification of color. CIENA is a simple, low-cost tool that could be applicable in various types of nucleic acid analyses, such as the quantification of nucleobase composition and the identification of damaged DNA.