DNA Shuffling, the In Vitro Molecular Evolution Technique, and Its Use in the Initial Pool Generation to Solve 26-Cities
TSP
Ji Youn LeeSchool of Chemical Engineering
Seoul National University
References• W. P. C. Stemmer, DNA shuffling by random fragmentation and reassembly I
n vitro recombination for molecular evolution Proc. Natl. Acad. Sci. USA (1994) 91 pp.10747~10751
• Fengzhu Sun, Modeling DNA shuffling
DNA Shuffling?!
selection
mutagenesis
amplification
In Vitro Evolution
Preparation of a pool of closely related molecules with different point mutations (through error-prone PCR or other mutation techniquessuch as oligonucleotide-directed mutagenesis).
DNA Shuffling
DNase I digestion
Sampling of fragments of lengths within a certain
range
PCR without added primers
Substrate preparation 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
10~ 30 ng/ ㎕ of purified fragments94℃ 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 primers1:40 dilution of the primerless PCR product into PCR mixture with 0.8 M each primer and ~15 additional cycles And… a single product of the correct size is typically obtained
Cloning and analysis
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.
reassembly analysis by sampling after 25, 30, 35, 40, and 45 cycles of reassembly
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?
vertexweight
edge
annealing
extension
denature
complementary vertex as a linker I species
0 W 1 W 2 W 3 W
1 1 W 2
0 to 1 1 to 2 2 to 3
1 to 2 2 to 3
c2
complementary (part of vertex+part of weight)As a linker II species
W+1
Thinking…- Complementary strand 의 존재로 인한 , self-hybridization- 만약 linker 를 20 mer 가 아닌 , 짧은 fragment 로 design 한다면 ? 10 mer 정도로 ..
