γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue
Alexander Orenstein,
April S. Berlyoung,
Elizabeth E. Rastede,
Ha H. Pham,
Elise Fouquerel,
Connor T. Murphy,
Brian J. Leibowitz,
Jian Yu,
Tumul Srivastava,
Bruce A. Armitage,
Patricia L. Opresko
Affiliations
Alexander Orenstein
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA
April S. Berlyoung
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA
Elizabeth E. Rastede
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA
Ha H. Pham
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA
Elise Fouquerel
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA
Connor T. Murphy
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA
Brian J. Leibowitz
Department of Pathology, University of Pittsburgh School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA
Jian Yu
Department of Pathology, University of Pittsburgh School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA
Tumul Srivastava
PNA Innovations, Inc., 10-N Roessler Rd., Woburn, MA 01801, USA
Bruce A. Armitage
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA
Patricia L. Opresko
Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA
Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.
