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Journal of Histochemistry and Cytochemistry, Vol. 47, 273-280, March 1999, Copyright © 1999, The Histochemical Society, Inc.

RAPID COMMUNICATION

In Situ Amplification Using Universal Energy Transfer-labeled Primers

G.J. Nuovoa, R.J. Hohmanb, G.A. Nardoneb, and I.A. Nazarenkob
a MGN Medical Research Laboratory, Setauket, New York
b Intergen Discovery Products, Gaithersburg, Maryland

Correspondence to: G.J. Nuovo, MGN Medical Res. Laboratory, 8 Huckleberry Lane, Suite 5, Setauket, NY 11733.

We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5' tail sequence, which is homologous to the 3' end of the UniPrimer which, in turn, has a hairpin structure on the 5' end. The hairpin structure brings the fluorophore and quencher into close proximity when the primer is free in solution, providing efficient quenching. When the primer is incorporated into the PCR product, the hairpin structure is unfolded and a fluorescent signal can be detected. Using hepatitis C and human papillomavirus as model systems, this study demonstrates several advantages in the hot-start in situ PCR technique with the UniPrimer system, including target specific detection of one DNA copy per cell without a separate in situ hybridization step and detection of an RNA target by RT in situ PCR without overnight DNase digestion. The UniPrimer-based in situ PCR allows rapid and simple detection of any DNA or RNA target without concern for the background from DNA repair invariably evident in paraffin-embedded tissue when a labeled nucleotide is used. (J Histochem Cytochem 47:273–279, 1999)

Key Words: in situ PCR, PCR, hepatitis C, HPV, HIV-1


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