Simple and Affordable Kinetic Assay of Nucleic Acids by Gel Staining

Abstract

In an effort to circumvent the pitfalls of using radioactive labeling to measure kinetics in undergraduate labs, a gel staining kinetics assay was developed. The IR‐3 enzyme and substrate, a single stranded DNA that cleaves another single stranded DNA only in the presence of Zn++, was used as a simple and inexpensive DNA model. The rate of the IR‐3, or D‐Zyme, was successfully determined by staining PAGE‐Urea gels with SYBR Gold and then viewing them at 302 nm with epi‐illumination. The open source software ImageJ (version 1.52K) was utilized to quantify the relative band intensities, the ratio of cleaved product to the addition of product and substrate, gave percent cleavage. When percent cleavage was plotted against time in Minitab (version 18.1, Minitab, Inc.), the rate of the reaction could then be determined by nonlinear regression curve‐fitting to the single exponential. To certify this technique, it was also tested on two other models, one being a modified version of the D‐Zyme, while the other was a known Hammer Head Ribozyme, trans RzB. It was demonstrated that the use of SYBR Gold post‐migration staining can be used to quantify RNA and DNA bands for cleavage kinetic analysis, without substrate labeling. Although this method is comparatively less sensitive than radioactive and fluorescence labeling, it has several advantages such as: i) ease of use; ii) cost effective; iii) no half‐life decay; iv) absence of substrate labeling; v) precision enabling curve‐fitting; vi) versatility with non‐structured and structured RNAs or DNAs.Support or Funding InformationMonmouth University Summer Research Program