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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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

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 Shuffling


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


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


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

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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