The use of immobilized caffeine aptamers in decaffeination & Roles of co-precipitants in the precipitation of DNA

Abstract

With the growing consumption of decaffeinated beverages, the development of eco-friendly and cost-effective methods to achieve decaffeination has been a growing field within the biotechnology industry. This thesis investigated the selection of a caffeine-specific aptamer through in-vitro selection. Using immobilized targets of caffeine and theophylline, and capped immobilized targets of caffeine and heptanoic acid, a library of caffeine-specific aptamers was obtained. Illumina sequencing was utilized to obtain sequences of the resulting aptamer pool with two sequences, N-12 and N-13-1, being selected for further characterization. Preliminary isothermal titration calorimetric experiments showed weak binding to caffeine. Future work will examine the binding of these two sequences and other potential sequencies in detail. DNA precipitation protocols are an integral part of many biotechnology and molecular biology laboratory procedures. When precipitating samples of low concentrations, co-precipitants are typically used to help facilitate the precipitation process. In this study, the validation of using co-precipitants in DNA precipitation protocols was conducted. Three carbohydrate-based co-precipitants, glycogen, dextran and β-glucan at concentrations of 20 mg/mL were tested to determine the optimal conditions for DNA recovery through precipitation with no clear improvements in DNA precipitation being reported. Additionally, the effects of co-precipitants on DNA duplex formation, as investigated using circular dichroism, was found to be minimal with no notable differences. Furthermore, the potential interferences of co-precipitants on B-DNA to Z-DNA transitions were investigated using circular dichroism. It was found that the use of co-precipitants does not have significant effects on Z-DNA transitions. On the other hand, some changes in the circular dichroism of B-DNA were observed in the presence of glycogen and dextran. Future work will explore these conformations changes.