Ji Youn Lee School of Chemical Engineering Seoul National University

1. 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

2. 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

3. DNA Shuffling?!

4. In Vitro Evolution selection 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). mutagenesis amplification

5. DNA Shuffling

6. 1 kb dsDNA PCR products derived from pUC18 (reomoval of free primers) Substrate preparation 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 DNase I digestion Sampling of fragments of lengths within a certain range Fragments of 10~50 bp were purified from 2% low meltin point agarose gels 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 without added 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 PCR with primers Cloning and analysis

7. 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 error-prone 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.

8. Its Application to the Initial Pool Generation

9. 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?!

10. An Estimate of Oligomer Cost

14. Disadvantages • I have no experience! • I have no advisor! • Is it possible in the real world?

15. How It Works?

16. complementary vertex as a linker I species vertex weight complementary (part of vertex+part of weight) As a linker II species edge 0 W 1 W 2 W 3 W 1 to 2 0 to 1 2 to 3 1 1 W 2 annealing 1 to 2 2 to 3 extension c2 denature W+1 • Thinking… • Complementary strand의 존재로 인한, self-hybridization • 만약 linker를 20 mer가 아닌, 짧은 fragment로 design한다면? 10 mer 정도로..