Journal Article

Generation of Human Immunodeficiency Virus–1 Long Terminal Repeat Reporter Genes by Rapid Polymerase Chain Reaction–Mediated Mutagenesis

Juan Wang, HangJun Lv, Ling Xu, Jin Yang, ZongXing Yang and NanPing Wu

in Laboratory Medicine

Published on behalf of American Society for Clinical Pathology

Volume 44, issue 3, pages 220-227
Published in print August 2013 | ISSN: 0007-5027
Published online September 2015 | e-ISSN: 1943-7730 | DOI: https://dx.doi.org/10.1309/LMB66MQ8JIXDCCQJ
Generation of Human Immunodeficiency Virus–1 Long Terminal Repeat Reporter Genes by Rapid Polymerase Chain Reaction–Mediated Mutagenesis

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  • Clinical Cytogenetics and Molecular Genetics
  • Molecular and Cell Biology
  • Molecular Biology and Genetics

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Objective:

The development of a human immunodeficiency virus (HIV)–1 promoter reporter system is critical for investigating transcription activity of the virus. We designed a modified overlap extension PCR method for single round site-directed mutagenesis of the long terminal repeat (LTR) segment of HIV-1.

Methods:

The procedure consists of overlap extension PCR, DpnI digestion, and product transformation. Wild-type and serial sequence deletion clones of HIV-1 LTR were constructed. Basal transcription activity was also measured.

Results:

The overall efficiency for obtaining the desired products was 53.2%. The reporter assay indicated the importance of the second NF-κB cis-element and surrounding regions in mediating the regulation of HIV-1 transcription.

Conclusion:

The technique we investigated is suitable for high-throughput functional studies of virus-host interaction.

Keywords: mutation; deletion; PCR; clone

Journal Article.  3591 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics ; Molecular and Cell Biology ; Molecular Biology and Genetics

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