Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Construction of semi-randomized gene libraries with weighted oligonucleotide synthesis and PCR


Randomized gene libraries may be constructed and screened to find novel candidates with particular functions, and the applications can range widely, from protein engineering to selecting new microRNAs. Here we describe a technique to construct gene libraries using semi-randomized weighted oligonucleotide synthesis and end-to-end ligation. This method makes it possible to search the combinatorial space around a particular nucleotide sequence for a greater number of positions than is possible with fully randomized oligonucleotides. As an alternative to full cassette construction, library mutations can also be introduced through 'round-the-world PCR' approaches. Construction of a randomized gene cassette and cloning can typically be achieved in 2 weeks. Therefore, these are rapid and convenient methods to generate successive generations of libraries for iterative selection and optimization.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Gene randomization with weighted primer synthesis.
Figure 2: Gene library construction by different methods.


  1. Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317 (1985).

    Article  CAS  Google Scholar 

  2. Winter, G., Griffiths, A.D., Hawkins, R.E. & Hoogenboom, H.R. Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455 (1994).

    Article  CAS  Google Scholar 

  3. Hanes, J. & Pluckthun, A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942 (1997).

    Article  CAS  Google Scholar 

  4. Lipovsek, D. & Pluckthun, A. In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51–67 (2004).

    Article  CAS  Google Scholar 

  5. Tawfik, D.S. & Griffiths, A.D. Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652–656 (1998).

    Article  CAS  Google Scholar 

  6. Campbell, T.B. & Cech, T.R. Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA. RNA 1, 598–609 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhao, H.F. et al. High-throughput screening of effective siRNAs from RNAi libraries delivered via bacterial invasion. Nat. Methods 2, 967–973 (2005).

    Article  CAS  Google Scholar 

  8. Isalan, M., Santori, M.I., Gonzalez, C. & Serrano, L. Localized transfection on arrays of magnetic beads coated with PCR products. Nat. Methods 2, 113–118 (2005).

    Article  CAS  Google Scholar 

  9. Isalan, M., Klug, A. & Choo, Y. A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat. Biotechnol. 19, 656–660 (2001).

    Article  CAS  Google Scholar 

  10. Griffiths, A.D. & Tawfik, D.S. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J. 22, 24–35 (2003).

    Article  CAS  Google Scholar 

  11. Scott, J.K. & Smith, G.P. Searching for peptide ligands with an epitope library. Science 249, 386–390 (1990).

    Article  CAS  Google Scholar 

  12. Tong, A.H. et al. A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Science 295, 321–324 (2002).

    Article  CAS  Google Scholar 

  13. Reynolds, L. et al. Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. Proc. Natl. Acad. Sci. USA 100, 1615–1620 (2003).

    Article  CAS  Google Scholar 

  14. Santori, M.I., Serrano, L. & Isalan, M. Transfection with magnetic beads coated with PCR products and other nucleic acids. Nat. Protocols 1, (2006) (doi:10.1038/nprot2006.74).

  15. del Rio, G., Osuna, J. & Soberon, X. Combinatorial libraries of proteins: analysis of efficiency of mutagenesis techniques. Biotechniques 17, 1132–1139 (1994).

    CAS  PubMed  Google Scholar 

  16. Hermes, J.D., Parekh, S.M., Blacklow, S.C., Koster, H. & Knowles, J.R. A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers. Gene 84, 143–151 (1989).

    Article  CAS  Google Scholar 

  17. Isalan, M., Klug, A. & Choo, Y. Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers. Biochemistry 37, 12026–12033 (1998).

    Article  CAS  Google Scholar 

  18. Schymkowitz, J. et al. The FoldX web server: an online force field. Nucleic Acids Res. 33, W382–W388 (2005).

    Article  CAS  Google Scholar 

  19. Innis, M.A. PCR Protocols: A Guide to Methods and Applications (Academic Press, San Diego, 1990).

    Google Scholar 

  20. Miyazaki, K. & Takenouchi, M. Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. Biotechniques 33, 1033–1034, 1036–1038 (2002).

    Article  CAS  Google Scholar 

  21. Kim, Y.G. & Maas, S. Multiple site mutagenesis with high targeting efficiency in one cloning step. Biotechniques 28, 196–198 (2000).

    Article  CAS  Google Scholar 

  22. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 1989).

    Google Scholar 

  23. Isalan, M. & Choo, Y. Rapid, high-throughput engineering of sequence-specific zinc finger DNA-binding proteins. Methods Enzymol. 340, 593–609 (2001).

    Article  CAS  Google Scholar 

  24. Papworth, C., Bauer, J.C. & Braman, J. Site-directed mutagenesis in one day with >80% efficiency. Strategies 9, 3–4 (1996).

    Article  Google Scholar 

Download references


The author would like to thank P. Beltrao for critical reading of the manuscript.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Mark Isalan.

Ethics declarations

Competing interests

The author declares no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Isalan, M. Construction of semi-randomized gene libraries with weighted oligonucleotide synthesis and PCR. Nat Protoc 1, 468–475 (2006).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing