Quantitative Fluorescence In Situ Hybridization (Q‐FISH)

This unit describes a quantitative technique for measuring the lengths of telomere repeat sequences in individual chromosomes from single metaphase cells. The technique is based on fluorescence in situ hybridization (FISH) adapted for use with peptide nucleic acid (PNA) probes. PNA is an example of novel synthetic oligonucleotide "mimetic" which has a higher affinity than regular oligonucleotide (RNA or DNA) probes for complementary single-strand (ss) DNA sequences. PNA oligonucleotides have excellent penetration properties due to their small size (typically 15 to 18-mers) and can be directly labeled with fluorochromes. These properties have been exploited to develop quantitative fluorescence in situ hybridization (Q-FISH) onto denatured single-stranded chromosomal DNA target sequences. The latter can be present in preparations of fixed metaphase cells on slides (Q-FISH) or in heat-treated (interphase) cells in suspension (flow-FISH).