γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue
Abstract
:1. Introduction
2. Results
2.1. Design of Telomeric γPNA FRET Pair Miniprobes
2.2. Hybridization of γPNA Miniprobes to DNA Oligonucleotides
2.3. Telomere Staining by FRET γPNA Miniprobes in Human Cells
2.4. FRET Miniprobes Exhibit Reduced Background Staining on Chromosome Spreads
2.5. Elimination of Wash Steps Improves qFISH Detection of Telomeres in Tissue Sections
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Thermal Melting Analysis
4.3. Circular Dichroism (CD) Spectroscopy
4.4. Fluorescence Spectroscopy
4.5. Cell Culture
4.6. Fluorescent In Situ Hybridization of Interphase Cells
4.7. Fluorescent In Situ Hybridization of Metaphase Chromosomes
4.8. Tissue Fluorescent In Situ Hybridization
4.9. Imaging
4.10. γPNA Fluorescence Intensity Measurements
4.11. Statistical Analyses
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sample Availability: Samples of the ©PNA FRET miniprobes are available from the authors. |
Name | Sequence (C-to-N for γPNA) |
---|---|
SCy3-γPNA | H2N–TCC CAA TCC–SCy3 |
X647-γPNA | H2N–CAA TCC CAA–X647 |
Telo-3 DNA | 5′–(AGG GTT)3–3′ |
Telo-4.5 DNA | 5′–(GTT AGG)4 GTT–3′ |
9-mer A DNA | 5′–AGG GTT AGG–3′ |
9-mer B DNA | 5′–GTT AGG GTT–3′ |
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Orenstein, A.; Berlyoung, A.S.; Rastede, E.E.; Pham, H.H.; Fouquerel, E.; Murphy, C.T.; Leibowitz, B.J.; Yu, J.; Srivastava, T.; Armitage, B.A.; et al. γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue. Molecules 2017, 22, 2117. https://doi.org/10.3390/molecules22122117
Orenstein A, Berlyoung AS, Rastede EE, Pham HH, Fouquerel E, Murphy CT, Leibowitz BJ, Yu J, Srivastava T, Armitage BA, et al. γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue. Molecules. 2017; 22(12):2117. https://doi.org/10.3390/molecules22122117
Chicago/Turabian StyleOrenstein, Alexander, April S. Berlyoung, Elizabeth E. Rastede, Ha H. Pham, Elise Fouquerel, Connor T. Murphy, Brian J. Leibowitz, Jian Yu, Tumul Srivastava, Bruce A. Armitage, and et al. 2017. "γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue" Molecules 22, no. 12: 2117. https://doi.org/10.3390/molecules22122117