Zhang MPP 2011
From MPPWiki
Optimizing Nucleic Acid Hybridization Specificity
- David Zhang
- Harvard University
Abstract
The specific hybridization of complementary sequences is an essential property of nucleic acids, and enables a wide variety of reactions and functions, both biological and biotechnological. However, the specificity of nucleic acid hybridization is compromised for longer strands, except near the melting temperature. Here, we present a general and rigorous analysis framework for evaluating the specificity of nucleic acid hybridization probes based on thermodynamics. Using this framework, we analytically derived the optimal discrimination factor achievable for a single-base change, and designed a hybridization probe that can achieve near-optimal discrimination factors by mimicking melting temperature hybridization conditions. We comprehensively tested our probes for 5 different DNA targets and 55 spurious analogs that contain energetically representative single-base changes (replacements, deletions, and insertions), and these probes produced discrimination factors between 3 and 200 (median 26). Without retuning, our probes function robustly across diverse conditions: temperature (10 $^\circ$C to 37 $^\circ$C), salinities (1.15 mM Mg$^{2+}$ to 47.2 mM Mg$^{2+}$), and oligonucleotide concentrations (100 nM to 1 $\mu$M). Experiments with RNA also showed effective single-base change discrimination.
