Displacement Whiplash PCR: Optimized Architecture and Experimental Validation
Whiplash PCR-based methods of biomolecular computation (BMC), while highly-versatile in principle, are well-known to suffer from a simple but serious form of self-poisoning known as back-hybridization. In this work, an optimally re-engineered WPCR-based architecture, Displacement Whiplash PCR (DWPCR) is proposed and experimentally validated. DWPCR’s new rule protect biostep, which is based on the primer-targeted strand-displacement of back-hybridized hairpins, renders the most recently implemented rule-block of each strand unavailable, abolishing back-hybridization after each round of extension. In addition to attaining a near-ideal efficiency, DWPCR’s ability to support isothermal operation at physiological temperatures eliminates the need for thermal cycling, and opens the door for potential biological applications. DWPCR should also be capable of supporting programmable exon shuffling, allowing XWPCR, a proposed method for programmable protein evolution, to more closely imitate natural evolving systems. DWPCR is expected to realize a highly-efficient, versatile platform for routine and efficient massively parallel BMC.
KeywordsKlenow Fragment Strand Displacement Template Strand Polymerization Experiment Prime Strand
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