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DNA Shuffling, the In Vitro Molecular Evolution Technique, and Its Use in the Initial Pool Generation to Solve 26-Cities TSP Ji Youn Lee School of Chemical Engineering Seoul National University References • W. P. C. Stemmer, DNA shuffling by random fragmentation and reassembly In vitro recombination for molecular evolution Proc. Natl. Acad. Sci. USA (1994) 91 pp.10747~10751 • Fengzhu Sun, Modeling DNA shuffling DNA Shuffling?! In Vitro Evolution selection mutagenesis amplification Preparation of a pool of closely related molecules with different point mutations (through error-prone PCR or other mutation techniques such as oligonucleotide-directed mutagenesis). DNA Shuffling Substrate preparation DNase I digestion Sampling of fragments of lengths within a certain range 1 kb dsDNA PCR products derived from pUC18 (reomoval of free primers) 2~4 ㎍ of the DNA substrate + 0.0015 unit of DNase I per ㎕ in 100 ㎕ of 50 mM Tris-HCl, pH 7.4, 1mM MgCls for 10~20 min at RT Fragments of 10~50 bp were purified from 2% low meltin point agarose gels PCR without added primers 10~30 ng/㎕ of purified fragments 94℃ for 1 min (94℃ for 0.5 min, 50~55 ℃ for 0.5 min and 72℃ for 0.5 min) 72℃ for 5 min PCR with primers 1:40 dilution of the primerless PCR product into PCR mixture with 0.8 mM each primer and ~15 additional cycles And… a single product of the correct size is typically obtained Cloning and analysis reassembly analysis by sampling after 25, 30, 35, 40, and 45 cycles of reassembly Results - When high concentration of fragments (10~30 ng/microliter) was used, the reassembly reaction was surprisingly reliable. - Reassembly process introduces point mutations at a rate of 0.7%, which is similar to errorprone PCR. - The rate of point mutagenesis may depend on the size of the fragments that are used in the reassembly. - In contrast to PCR, DNA reassembly is an inverse chain reaction. Its Application to the Initial Pool Generation Advantages • More economic! – – – – No need of phosphorylation No need of ligase (terrible labour of course…) dNTPs are much cheaper than oligomers We can use the saved money for the study of bead separation • More reliable! – No need of hybridization/ligation step – Lower concentration of the initial olgomers is tolerable?! – We believe the potential of PCR • Originality?! An Estimate of Oligomer Cost Disadvantages • I have no experience! • I have no advisor! • Is it possible in the real world? How It Works? complementary vertex as a linker I species vertex weight complementary (part of vertex+part of weight) As a linker II species edge 0 annealing W 0 to 1 1 1 W 1 to 2 2 W 2 to 3 1 1 to 2 3 W W 2 extension denature c2 W+1 Thinking… - Complementary strand의 존재로 인한, self-hybridization - 만약 linker를 20 mer가 아닌, 짧은 fragment로 design한다면? 10 mer 정도로.. 2 to 3