Characterization of Non-Crosshybridizing DNA Oligonucleotides Manufactured In Vitro

1
Characterization of Non-Crosshybridizing DNA
Oligonucleotides Manufactured In Vitro

• J. Chen, R. Deaton, M. Garzon, J. W. Kim, D.
  Wood, H. Bi, D. Carpenter, Y.-Z. Wang
• DNA10, pp. 132-141
• 2004. 6. 29.
• MEC Seminar
• Summarized by In-Hee Lee

2
Introduction

• In vitro protocol for generating
  non-crosshybridizing DNA oligonucleotides.
• Refer to DNA8 proceedings.

3
Introduction

• Advantages
• The oligonucleotides are selected in the
  conditions under which computations will be done.
• The potential for very large libraries.
• Prove the non-crosshybridizing characteristics of
  the library experimentally.

4
Cloning and Sequencing of Library Oligonucleotides
Cycle
Start 2(2) 20(20)
1 8(3) 22(8.3)
2 12(4) 33(11)
3 5(3) 23.8(14.3)
4 6(3) 28.6(14.3)
Decrease in the of stable crosshybridization(?)
5
Library Characterization with Gels

• Its intensity means the number of extended
  products.
• The intensity increase with cycle.
• More product is amplified over the cycle.

6
Library Characterization with Gels

• No self-hybridization in top and bottom strands.

7
Library Characterization with Spectroscopy

• Melting curve investigation
• Protocol product
• Random DNA samples of length 20.
• As a starting population of the protocol.
• 40 non-crosshybridizing oligonucleotides of
  length 20 Deaton at al., 2003
• Single-stranded oligonucleotide of length 20.
• 3 4 as a standard for non-crosshybridization.
• Watson-Crick pair of 2 sequences from 3.
• As a standard for hybridization.

8
Random seq.
Top strands from cycle 4.
NCH sequences
Top and bottom strands from cycle 4.
WC complements
9
Conclusion

• The experimental result indicate that the
  protocol was selecting for populations of
  non-crosshybridizing sequences.