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A DNA-based in vitro Genetic Program

This paper presents innovative methodologies in DNA-based in vitro genetic programming, focusing on the implementation of PWPCR and PNA-mediated Whiplash PCR. It describes a system of autonomous finite state machines that execute generative and search strategies while addressing the exponential size of solution spaces. By incorporating evolutionary strategies, particularly the Evolutionary PWPCR (EWPCR), the study showcases methods to evaluate fitness, perform selection and recombination, and generate restriction sites. The results aim to enhance the efficiency and effectiveness of synthetic biology applications.

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A DNA-based in vitro Genetic Program

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  1. A DNA-based in vitro Genetic Program J.A. Rose, M. Hagiya, R.J. Deaton, A. Suyama Journal of Biological Physics, 2002

  2. Introduction • PWPCR • PNA-mediated Whiplash PCR • Autonomous, parallel execution of a set of finite state machines • Generate and Search Strategy • Limited by the exponential size of the solution space Combination with Evolutionary Strategy Evolutionary PWPCR (EWPCR) to evolve approximate solutions to HPP

  3. PNA-Mediated Whiplash PCR Reduce Back-Hybridization!

  4. A PWPCR-based in vitro Genetic Program for HPP Vertexstate, Edgestate transition r: restriction site BseDI(CCTTGG) Gi: block’s initial state and relative order from the 5’ end P1, P2: primer for PCR rr: restriction site SnaBI(TACGTA) for 5’ de-biotinylation R: restriction site EcoRV(GATATC) for 3’ tail detachment

  5. EWPCR • Initialization • Fitness Evaluation • Selection • Recombination • Re-Initialization

  6. Initialization • Combinatorial incomplete mixture of rule blocks.

  7. Fitness Evaluation Eleminate haripin structure Prevent unexpected annealing Genereate a restriction site at rr T1 Tq q-1 affinity separation If Tq is nonempty or if Tq is empty and gmax then terminate and assign yes/no

  8. Selection • Fitness-squared-proportional selection • Restriction of all strands at R • Remove 3’ tail • For each tube Tf • PCR amplified • -rrP1, and RP’2 (- : biotinylation) • Vf = n0f2/Cfb is merged to form new tube Ts • b = sum of all f2 • Cf = concentration of tube Tf • Reanchored, denatured and washed

  9. Recombination • Ts is divided into Tc and Tc’ • With volume Vc = pcVs and Vc’ = Vs-Vc’ • Pc crossover probability • Tc  recombinationTc’ bypass recombination • Tc is divided into q-1 tubes of equal volume • Tj: j = 1~q-1 • Uniform-length, single point, two-parent crossover at rule block i=j • Primer extension by primer Gj’ • Restriction and Recombination

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