A method to accurately quantitate intensities of (32)P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh.

Abstract

Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs). Following imaging, multiple (32)P-bands are often present in lanes. Though individual bands appear by eye to be simple and well-resolved, scanning reveals they are actually skewed-Gaussian in shape and neighboring bands are overlapping, which complicates quantitation, because slower migrating bands often have considerable contributions from the trailing edges of faster migrating bands. A method is described to accurately quantitate adjacent (32)P-bands, which relies on having a standard: a simple skewed-Gaussian curve from an analogous pure, single-component band (e.g., primer alone). This single-component scan/curve is superimposed on its corresponding band in an experimentally determined scan/curve containing multiple bands (e.g., generated in a primer-extension reaction); intensity exceeding the single-component scan/curve is attributed to other components (e.g., insertion products). Relative areas/intensities are determined via pixel analysis, from which relative molarity of components is computed. Common software is used. Commonly used alternative methods (e.g., drawing boxes around bands) are shown to be less accurate. Our method was used to study kinetics of dNTP primer-extension opposite a benzo[a]pyrene-N(2)-dG-adduct with four DNAPs, including Sulfolobus solfataricus Dpo4 and Sulfolobus acidocaldarius Dbh. Vmax/Km is similar for correct dCTP insertion with Dpo4 and Dbh. Compared to Dpo4, Dbh misinsertion is slower for dATP (∼20-fold), dGTP (∼110-fold) and dTTP (∼6-fold), due to decreases in Vmax. These findings provide support that Dbh is in the same Y-Family DNAP class as eukaryotic DNAP κ and bacterial DNAP IV, which accurately bypass N(2)-dG adducts, as well as establish the scan-method described herein as an accurate method to quantitate relative intensity of overlapping bands in a single lane, whether generated from (32)P-signals or by other means (e.g., staining).