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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