1. Ji Youn Lee School of Chemical Engineering Seoul National University
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 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).
mutagenesis
amplification

5. DNA Shuffling

6. Substrate preparation
1 kb dsDNA PCR products derived from pUC18
(reomoval of free primers)
Substrate preparation
DNase I digestion
2~4 ㎍ of the DNA substrate unit of DNase I per ㎕ in 100 ㎕ of 50 mM Tris-HCl, pH 7.4, 1mM MgCls for 10~20 min at RT
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
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

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! More reliable! Originality?!
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. vertex
complementary vertex as a linker I species
weight
complementary (part of vertex+part of weight)
As a linker II species
edge W 1 W 2 W 3 W
0 to 1
1 to 2
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..