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Journal of Histochemistry and Cytochemistry, Vol. 48, 285-294, February 2000, Copyright © 2000, The Histochemical Society, Inc.

TECHNICAL NOTE

PCR-derived ssDNA Probes for Fluorescent In Situ Hybridization to HIV-1 RNA

Marlyse C. Knuchela, Brigit Grafa, Erika Schlaepfera, Herbert Kustera, Marek Fischera, Rainer Webera, and Richard W. Conea
a Division of Infectious Diseases, Department of Internal Medicine, University Hospital Zurich, Zurich, Switzerland

Correspondence to: Marlyse C. Knuchel, University Hospital, Div. of Infectious Diseases, Raemistr. 100, CH-8091 Zurich, Switzerland.

We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 µg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285–293, 2000)

Key Words: fluorescent in situ hybridization, ssDNA probes, RNA probes, HIV-1


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