DNA Shuffling, the In Vitro Molecular Evolution Technique, and Its Use in the Initial Pool Generatio...
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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.

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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


References

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

DNA Shuffling?!


In vitro evolution

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


Dna shuffling1

DNA Shuffling


Ji youn lee school of chemical engineering seoul national university

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


Ji youn lee school of chemical engineering seoul national university

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.


Its application to the initial pool generation

Its Application to the Initial Pool Generation


Advantages

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

An Estimate of Oligomer Cost


Disadvantages

Disadvantages

  • I have no experience!

  • I have no advisor!

  • Is it possible in the real world?


How it works

How It Works?


Ji youn lee school of chemical engineering seoul national university

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..


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