DNA Sequence Context and Multiplex Hybridization Reactions:  Melting Studies of Heteromorphic Duplex DNA Complexes

Abstract

Heteromorphic hybrid duplex DNA complexes are duplex states, other than perfectly matched duplexes, that can form when single strands comprising several different perfectly matched duplexes are simultaneously present in solution. Such cross-hybridization "side reactions" are of particular nuisance in multiplex reaction schemes, where many strands are designed to hybridize in parallel fashion with only their corresponding perfect complement strand. Relative to the perfect match duplexes, the sequence dependent features of these heteromorphic duplex states and their thermodynamic stability are an important consideration for multiplex hybridization reaction design. We have measured absorbance versus temperature melting curves and performed differential scanning calorimetry measurements on various mixtures of eight different 24 base single strands. When perfect complementary pairs of strands are mixed in single reactions, four perfectly matched duplexes form. When mixtures of strands that are not perfectly matched are prepared and analyzed, melting transitions for cross-hybridization are observed along with significant hyperchromicity changes. This is indicative of a melting hybrid, heteromorphic duplex states formed from two nonperfectly matched strands. In addition, when both the perfectly matched and noncomplementary strands are mixed together (in multiplex hybridization reactions) at molar ratios of 1:1, 3:1, and 1:3, evidence of perfect duplex and heteromorphic duplex complexes is found in all cases. A new analytical tool for considering heterogeneous, duplex complexes in multiplex hybridization mixtures is presented and employed to interpret the acquired melting data.

AutoDimer: a screening tool for primer-dimer and hairpin structures

“…These parameters are intended for calculating the thermodynamic effect of a single base pair mismatch. Complexities exist due to multiple mismatches and mismatch context within a duplex (33,34). Due to these issues, a conservative approach was implemented for dealing with mismatched base pairing.…”

Statistical Thermodynamics and Kinetics of DNA Multiplex Hybridization Reactions

“…Exceptions are studies of solutions containing a single species of a linear or circular self-complementary single strand for the purpose of evaluating DNA sequence-dependent stability parameters, and examining intramolecular hairpin stability (43)(44)(45), and studies performed for the purpose of investigating the thermodynamic parameters of single basepair mismatches (1,9,46), bulged loops (47)(48)(49), or multiple strand mixtures, such as triplexes and quadruplex (50). Melting studies of mixtures of more than two strands in solution, which can form both perfect match and mismatch duplex complexes, have suggested that sequence-dependent interactions in tandem mismatches might contribute to the stability of mismatch hybrid duplex complexes (51). For this and other reasons described herein, when multiple strands meant to form perfectly matched duplexes are present together in a reaction mix, it cannot be presumed that each pair of strands will form the appropriate duplex, exclusively, in the same way they would in isolation, in the absence of the influence from other single strands (and duplexes).…”

Nanomechanical Detection of DNA Melting on Microcantilever Surfaces

“…Given that they are driven by free energy reductions that usually involve enthalpy changes, they are often detected by calorimetric measurements . Differential scanning calorimetry has shown to effectively measure the melting temperature ( T m ) and enthalpy of homogeneous and heterogeneous DNA strands have been developed for multiplexed calorimetry, thermal insulation of a calorimeter from its surroundings becomes increasingly difficult for small samples where the surface-to-volume ratio increases.…”