Abstract
Sensitive and specific DNA hybridization is essential for nucleic acid chemistry. Competitive composition of probe and blocker has been the most adopted probe design for its relatively high sensitivity and specificity. However, the sensitivity and specificity were inversely correlated over the length and concentration of the blocker strand, making the optimization process cumbersome. Herein, we construct a theoretical model for competitive DNA hybridization, which disclose that both the thermodynamics and kinetics contribute to the inverse correlation. Guided by this, we invent the 4-way Strand Exchange LEd Competitive DNA Testing (SELECT) system, which breaks up the inverse correlation. Using SELECT, we identified 16 hot-pot mutations in human genome under uniform conditions, without optimization at all. The specificities were all above 140. As a demonstration of the clinical practicability, we develop probe systems that detect mutations in human genomic DNA extracted from ovarian cancer patients with a detection limit of 0.1%.
Highlights
Sensitive and specific DNA hybridization is essential for nucleic acid chemistry
The results demonstrated the 4-way Strand Exchange LEd Competitive DNA Testing (SELECT) system could break up the inverse correlation between sensitivity and specificity
For the 4-way SELECT system, the assumption only required that bonding of blocker to target is strong (KBT ≫ 0), and the blocker did not need to outcompete probe because the competition between them was eliminated by the formation of BS
Summary
Sensitive and specific DNA hybridization is essential for nucleic acid chemistry. Competitive composition of probe and blocker has been the most adopted probe design for its relatively high sensitivity and specificity. Numerous nucleic acid assays have been developed for discrimination of single-base mismatches including probes with complex structures[18,19], enzyme assisted DNA probes[20,21], barcode assays[22,23], selective PCR24,25 and next-generation sequencing[26,27] All of these methods rely on the specificity of DNA hybridization at some steps of their working procedures. As reported in previous literatures, the sensitivity of the competitive composition systems was in reverse correlation to the specificity when changing the length and concentration of blocker, which meant higher sensitivity led to lower specificity and vice versa[32] This intrinsic contradiction resulted in labour intensive optimization of the structure and concentration of the blocker strand and probe. High-throughput analysis of multiple mutations[35,36,37], which required design of multiple compositions of blocker strand and probe, was very difficult and time-consuming under current DNA probe systems
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