Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, UPMC Hillman Cancer Center, Pittsburgh, PA 15213, USA.
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Molecules. 2017 Dec 2;22(12):2117. doi: 10.3390/molecules22122117.
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.
荧光原位杂交法测量端粒长度被广泛应用于生物医学和流行病学研究，但自首次报道以来，该技术在 20 年里的发展相对较少。本报告介绍了使用双 γPNA（γPNA）探针的方法，这些探针在沿着端粒的交替位置杂交，并产生Förster 共振能量转移（FRET）信号。在细胞核、染色体铺片和组织样本中观察到端粒的明亮染色。FRET 检测的使用还允许消除通常需要的洗涤步骤，以去除会导致背景信号的未杂交探针。我们发现，这些洗涤步骤会通过去除结合的和未结合的探针而降低信号强度，因此消除这些步骤不仅可以加速该过程，还可以提高染色质量。因此，γPNA FRET 对允许在广泛的研究和临床格式中更亮、更快地染色端粒。
