DNA sequence context and multiplex hybridization reactions: melting studies of heteromorphic duplex DNA complexes

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.