Determination of the Binding Constant between Oligonucleotide-Coupled Magnetic Microspheres and Target DNA

Abstract

Oligonucleotide-coupled magnetic microspheres (PMPs) are commonly used as solid carriers in genetic analysis to specifically recognize, capture, and manipulate target DNA. Determining the binding constant (K A ) of nucleic acid hybridization using an assay based on magnetic microspheres (MPs) and evaluating the performance of magnetic microspheres as solid carriers has significance for many applications. In this work, we established a simple doublereciprocal plot method to determine the binding constant of nucleic acid hybridization based on magnetic microspheres. The main experimental parameters were first optimized, and the binding constants between PMPs with single-stranded DNA (ssDNA) and PMPs with double-stranded DNA (dsDNA) were 1.07 ± 0.01 × 10 8 and 0.77 ± 0.02 × 10 8 M −1 , respectively, through the established double-reciprocal plot method. Oligonucleotidecoupled magnetic microspheres have the same initial effective target binding sites (A 0 ), regardless of whether the target molecules are single-stranded or double-stranded. In addition, the binding constants between PMPs with ssDNA and PMPs with dsDNA were 18.58 and 13.37%, respectively, of the binding constants measured in solution by isothermal titration calorimetry. This finding indicates that the binding constant based on solid phase hybridization is not significantly lower than that of liquid phase hybridization, even in the presence of competing target nucleic acid molecules in solution.

Electrochemical Beacon Method to Quantify 10 Attomolar Nucleic Acids with a Semilog Dynamic Range of 7 Orders of Magnitude

“…We conjecture that the lower LOQ was due to poor binding efficiency owing to the strong adsorption of the probe to the Au electrode due to the high G−C fraction (Figure 2c). To quantify the change in binding efficiency on the G−C fraction, unfortunately, the binding constant, unlike for fluorescence-based systems, 48 is difficult primarily because binding to probes that are up will lead to no change in the signal, while the ones down will lead to a change in the signal [as shown in Figure 5(iii) and experimentally shown in Figure 6a]. To independently measure the binding constant for up versus down probes that will be different requires concomitant measurement of the structure in real-time to follow the kinetics.…”

Molecular genetic methods for identifying raw materials in meat products: Diversity, opportunities and prospects

“…The demand for nucleic acids as detectable targets is determined by the combination of conservative and varying (including species-specific) sectors within their structure, as well as the possibility to implement high-affinity interactions with complementary oligonucleotides, thereby ensuring high specificity of analytical methods [13,14]. DNA is present in all animal tissues and features very high stability when exposed to high temperatures during the production of meat food products [11].…”