SpringerProtocols: Abstract: 9. PNA-In Situ Hybridization Method for Detection of HIV-1 DNA in Virus-Infected Cells and Subsequent Detection of Cellular and Viral Proteins

9. PNA-In Situ Hybridization Method for Detection of HIV-1 DNA in Virus-Infected Cells and Subsequent Detection of Cellular and Viral Proteins

By: Tomoko Hagiwara, Junko Hattori, Tsuguhiro Kaneda

Abstract

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We describe in situ hybridization protocols using peptide nucleic acid (PNA) as a probe for detecting HIV-1 DNA in virus-infected cells and the subsequent detection of cellular and/or viral proteins. Because a PNA probe of approx 20 bases was sufficiently long to detect a specific target sequence, a conserved sequence of such a short length was easily identified. Therefore, this probe is valuable even to identify quasi-species of HIV-1. In addition, we adopted a catalyzed signal amplification method to amplify weak viral DNA signals; thus, stringent washing was crucial for eliminating false-positive signals. Our double-staining method using PNA-in situ hybridization and subsequent immunostaining enabled the active and inactive proviruses to be distinguished.

Book Title: In Situ Hybridization Protocols

Series: Methods in Molecular Biology | Volume: 326 | Pub. Date: Dec-15-2005 | Page Range: 139-149 | DOI: 10.1385/1-59745-007-3:139

Subject: Cell Biology

Key Words: In situ hybridization - peptide nucleic acid - catalyzed signal amplification - HIV-1 provirus - CD4-positive T lymphocytes - p24 - HLA-DR

References

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