Telomeres play critical roles in the maintenance of chromosomal stability, as well in replicative capacity of cells. These highly conserved repeat complex consist of (TTAGGG)n and (CCCTTA)n in every human chromosome . These telomeric structure are subject to shortening during cell division, it provides important biological clues in the studies of aging, cell senescence, cell replication, cell immortality, and transformation to cancer (1-6).
Recently, Landdorp and colleagues (7) developed a PNA -based fluorescence in situ hybridization (FISH) method for quantitative telomere length measurement. This peptide nucleic acid (PNA) probes having unique traits of neutral backbone, which allows for PNA/DNA binding to occur more rapidly and more tightly than DNA/DNA binding. Moreover, PNA probes can bind to DNA under low ionic strength conditions that disfavor reannealing of complimentary genomic strands. This advantage is particularly important for in situ hybridization experiments that target repetitive sequences, because both the length and the repetitive nature of the target sequences will affectively favor renaturation over hybridization with labeled probes. The high affinity of PNA probes to DNA also constitutes an important feature for chromosomal analysis. As studies have demonstrated that PNA probes could discriminate between two centromeric DNA repeats that differed by only a single base pair 8, 9 whereas standard FISH probes are unable to discriminate sequences with a single base resolution. PNA-FISH assay requires very few cells (~10 to 15), thus, small tissue samples including clinical biopsies, can be easily accommodated. In addition, the cells under study need not be actively cycling and there is no requirement for tissue disaggregation or cell culture. This method provides a more accurate assessment of telomere lengths than Southern blotting because confounding contributions from undesired cell types within tissue samples. |
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| Materials: |
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| PNA Telomere Probes |
| 1. |
PNA Telomere C or G probe can be purchase from BSI at 5 nmole or 10 nmole size |
| 2. |
PNA is dissolved in 100% deionized formamide concentration of either 10 µM working solution) or 100 µM (stock) |
| 3. |
Store PNA solution in polypro tubes in the dark at 4°C or at 20°C. PNA is stable in formamide for at least one years. |
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| II Required Solutions/Reagents |
PFS (phosphate buffered Saline)
Ethanol (70%, 80%, 90% and 100%)
37% formaldehyde
100% formamide
Blocking reagent, Cat# 1096176 (Roche dianostics)
DAPI I solution in antifade, Cat#32-804830 (Vysis)
PI solution in antifade, Cat#32-8004829 (Vysis) |
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| Telomeric FISH with PNA probes |
| 1. |
Prepare the metaphase chromosomes by following standard cytogenic procedure use of freshly prepared slides is highly recommended 10. |
| 2. |
Fix slides in methanol: acetic acid (3:1) for 1 to 2 hrs at room temperature, air dry, immerse in PBS for 5 min. and fix in 4% formaldehyde in PBS, pH 7.2 for 1.5-2 min. |
| 3. |
After fixation, the slide are dehydrated using a series of cold ethanol washes (70%, 80%, 90% and 100%) and air dried for 15 min. |
| 4. |
To each slide add 15 to 20 µl of hybridization mixture containing 100 nM of labeled PNA probe in 70% formamide, 1% (wt/vol.) blocking reagent in 10 mM Tris, pH 7.2. Cover the area with a coverslip and seal with rubber cement. Denature the DNA on the slide again at 80°C. for 3 min. followed by hybridization in the dark at 37°C. in wet chamber for 2 hrs. |
| 5. |
After hybridization, excessive exposure to light should be avoided to prevent fluorescent bleaching. The slides are washed three times with 70% formamide/10mM Tris pH 7l.2 for 10 min. and with TNT (0.05 M Tris/0.15 M NaCl/0.05% Tween-20, pH 7.5) for 5 min. Slides are dehydrated in the same ethanol series as previously used and dried in the dark. |
| 6. |
Chromosomes are counter-stained with either 01 µg/ml of DAPI in antifade for rhodamine and fluorescein labeled probes or 0.6 µg/ml of PI in antifade for fluorescein labeled probles. |
| 7. |
Slides can be stored at -20°C in the dark with cover slip for two weeks. |
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| Reference: |
| 1. |
de Lange T, Science 1998, 279:334-335 |
| 2. |
DePinho RA, Nature 2000, 408:248-254 |
| 3. |
Greider CW, Curr Biol 1998, 8:R178-R181 |
| 4. |
Harley CB, Villeponteau B, Curr Opin Genet Dev 1995, 5:249-255 |
| 5. |
Shay JW, Mol Med Today 1995, 1:378-384 |
| 6. |
Blackburn EH, Nature 1991, 350:569-572 |
| 7. |
Landorp et al, Human Mol. Genetics 1996, 5:685-691 |
| 8. |
Chen et a, Mann Genome 1999, 10:13-18. |
| 9. |
Chen et al, Mamm Genome 2000, 11:384-391. |
| 10. |
Lawrence et al. Science 1990 249:928-932 |
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