Advertisement

Analysis of Shuffled Libraries by Oligonucleotide Probe Hybridization

  • Peter Meinhold
  • John M. Joern
  • Jonathan J. Silberg
Part of the Methods in Molecular Biology™ book series (MIMB, volume 231)

Abstract

In vitro recombination is often used to generate genetic diversity for directed evolution. Recombination techniques that rely on fragment hybridization yield libraries with preferred crossover positions and, in some cases, a bias toward incorporation of one parent over the others. This limits the diversity, and thus the utility of the library. These biases vary depending on the technique used for recombination, the distribution of sequence similarities within the parental genes, and the efficiency by which the different parental genes are PCR amplified. To assess the diversity generated in a library of chimeras, sequences of a large number of chimeras are required. While DNA sequencing can yield this information, sequencing these genes is prohibitively expensive, especially when the genes being recombined are large. Oligonucleotide probe hybridization, in contrast, offers a cost-effective approach for obtaining information about library biases that allows for optimization of shuffling procedures. When coupled with functional information, this technique can provide information about the relationship between sequence and function (1,2).

Keywords

Sodium Dodecyl Sulfate Probe Site Identical Region Hybridization Oven Terminal Transferase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Joern, J. M., Meinhold, P., and Arnold, F. H. (2002) Analysis of shuffled gene libraries. J. Mol. Biol. 316, 643–656.PubMedCrossRefGoogle Scholar
  2. 2.
    Abecassis, V., Pompon, D., and Truan, G. (2000) High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Res. 28, E88.PubMedCrossRefGoogle Scholar
  3. 3.
    Sambrook, J. and Russell, D. W. (2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  4. 4.
    Schutz, E. and von Ahsen, N. (1999) Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches. Biotechniques 27, 1218–1224.PubMedGoogle Scholar
  5. 5.
    Tatusova, T. A. and Madden, T. L. (1999) BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol. Lett. 174, 247–250.PubMedCrossRefGoogle Scholar
  6. 6.
    Engels, W. R. (1993) Contributing software to the internet: the Amplify program. Trends Biochem. Sci. 18, 448–450.PubMedCrossRefGoogle Scholar
  7. 7.
    Darling, D. C. and Brickell, P. M. (1994) Nucleic Acid Blotting, Oxford University Press, New York, NY.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Peter Meinhold
    • 1
  • John M. Joern
    • 1
  • Jonathan J. Silberg
    • 1
  1. 1.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadena

Personalised recommendations