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 ..