Q-FISH protocol-PANAGENE.pdf

Protocol of PNA Telomere probe for FISH (Fluorescence in situ hybridization) Telomeres have important functions in the stability and replication of chromosomes. These functions are mediated by highly conserved repeats which consist of (TTAGGG)n in all vertebrates. The number of telomeric repeats in human somatic cells appears to decrease with cell divisions and with age. Telomere shortening may act as a mitotic clock in normal somatic cells and high levels of the enzyme telomerase (capable of elongating telomeres) have been found in tumor cells. The most commonly used tool to estimate telomere length is southern analysis of genomic DNA digested with selected restriction enzymes. Such analysis requires thousands of cells and provides only a crude estimate of the average number of TTAGGG repeats in the chromosomes of all cells analyzed. In principle, fluorescence in situ hybridization (FISH) should be able to provide information on the telomere length of individual chromosomes. Directly labeled oligonucleotide probes are attractive probes for such analysis because of their small size (good penetration properties), single strand nature (no denaturation of probe) and controlled synthesis. However the efficiency of oligonucleotide hybridizations for telomeric repeats has not been sufficient to extend this approach beyond qualitative studies of TTAGGG repeat sequences in chromosomes of various species. Recently, it was shown that peptide nucleic acid (PNA) oligonucleotide probes will hybridize with complementary oligonucleotide sequences and that the resulting duplexes are more stable than DNA/DNA or DNA/RNA duplexes. In PNA, the charged phosphate-(deoxy) ribose backbone of conventional DNA and RNA oligonucleotides is replaced by an uncharged backbone of repeating N-(2-amino ethyl)-glycine units linked by peptide bonds. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibilit