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Generation of random mutant libraries with multiple primers in a single reaction.

Publication ,  Journal Article
Lu, X; Hora, B; Cai, F; Gao, F
Published in: J Virol Methods
August 2010

Characterization of multiple sites in a single gene that are important in biological phenotypes is challenging due to the difficulty to generate many mutants representing all or a majority of combinations of mutations in the gene. Using the HIV-1 env and pol genes as templates, four random libraries were generated representing different combinations of mutations introduced by up to 36 mutagenesis primers in a single assay. Over 86% of the clones contained mutations and the mutants tended to have single or fewer mutations in the libraries. When protein size was used as a screening marker, all identified clones contained at least 2 mutations and up to 12 mutations were detected in a single clone. Nearly all mutant clones in each library contained unique mutations, indicating that mutants in the library were generated at random. Closely related mutations which were overlapped by neighboring mutagenesis primers were often introduced in this system. Analysis of the env library showed that some potential N-linked glycosylation sites did not increase the Env molecular mass significantly, suggesting they were not used for glycosylation or only limited carbohydrate moieties were added at these sites. This novel method can serve as a powerful tool to study the biological phenotypes of genes whose functions are determined by multiple sites.

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Published In

J Virol Methods

DOI

EISSN

1879-0984

Publication Date

August 2010

Volume

167

Issue

2

Start / End Page

146 / 151

Location

Netherlands

Related Subject Headings

  • pol Gene Products, Human Immunodeficiency Virus
  • env Gene Products, Human Immunodeficiency Virus
  • Virology
  • Polymerase Chain Reaction
  • Mutant Proteins
  • Mutagenesis, Site-Directed
  • HIV-1
  • Gene Library
  • DNA Primers
  • 3207 Medical microbiology
 

Citation

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Lu, X., Hora, B., Cai, F., & Gao, F. (2010). Generation of random mutant libraries with multiple primers in a single reaction. J Virol Methods, 167(2), 146–151. https://doi.org/10.1016/j.jviromet.2010.03.025
Lu, Xiaozhi, Bhavna Hora, Fangping Cai, and Feng Gao. “Generation of random mutant libraries with multiple primers in a single reaction.J Virol Methods 167, no. 2 (August 2010): 146–51. https://doi.org/10.1016/j.jviromet.2010.03.025.
Lu X, Hora B, Cai F, Gao F. Generation of random mutant libraries with multiple primers in a single reaction. J Virol Methods. 2010 Aug;167(2):146–51.
Lu, Xiaozhi, et al. “Generation of random mutant libraries with multiple primers in a single reaction.J Virol Methods, vol. 167, no. 2, Aug. 2010, pp. 146–51. Pubmed, doi:10.1016/j.jviromet.2010.03.025.
Lu X, Hora B, Cai F, Gao F. Generation of random mutant libraries with multiple primers in a single reaction. J Virol Methods. 2010 Aug;167(2):146–151.
Journal cover image

Published In

J Virol Methods

DOI

EISSN

1879-0984

Publication Date

August 2010

Volume

167

Issue

2

Start / End Page

146 / 151

Location

Netherlands

Related Subject Headings

  • pol Gene Products, Human Immunodeficiency Virus
  • env Gene Products, Human Immunodeficiency Virus
  • Virology
  • Polymerase Chain Reaction
  • Mutant Proteins
  • Mutagenesis, Site-Directed
  • HIV-1
  • Gene Library
  • DNA Primers
  • 3207 Medical microbiology