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Abstract
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.
Highlights
Telomeres are the specialized structures at the ends of linear chromosomes
We examined Förster resonance energy transfer (FRET) pair γPNA staining under similar fluorescence in situ hybridization (FISH) conditions, except we added back the traditional wash steps to remove unhybridized probes
9-mer γPNAs, designed hybridize in alternating theenables length of telomeric. This allowed γPNAs, designed to hybridize in alternating fashion along the length of telomeric. This allowed effective and quantitative telomere staining by the FRET miniprobes on fixed nuclei, metaphase effective and quantitative telomere staining by the FRET miniprobes on fixed nuclei, metaphase chromosomes, and fixed tissue sections from a variety of sources, including telomerase positive and chromosomes, and fixed tissue sections from a variety of sources, including telomerase positive and negative cell lines, and cells with very short telomeres
Summary
Telomeres are the specialized structures at the ends of linear chromosomes. They consist of tandem TTAGGG repeats, coated with the protein complex shelterin [1]. Telomere restriction fragment analysis by Southern blot has long been a standard in the field This method normally requires one to three micrograms of intact genomic DNA and only provides an average telomere length for a population of chromosomes and cells [10]. Quantitative fluorescence in situ hybridization (Q-FISH) using peptide nucleic acid (PNA, Chart 1) probes is the only available method for quantifying the shortest telomeres in intact cells and solid tissue [8]. Since most endogenous fluorophores have small Stokes shifts, fluorescence from a large Stokes shift dye will be red-shifted away from autofluorescence from the endogenous fluorophore, thereby reducing the background signal [24] This does not address the need for harsh denaturing and washing conditions associated with telomere FISH probes.
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