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PNA FISH Probes

Oligonucleotides

PNA* (Peptide Nucleic Acid) an artificially created DNA analogue was invented by Drs. Nielsen, Egholm, Berg, and Buchardt in 1991. The uncharged backbone of PNA suppresses the charge repulsion between PNA and the DNA/RNA strand.

In comparison with DNA Oligonucleotides, PNA oligomers demonstrate a higher sequence specificity, improved stability, reproducibility and lower background.


Please Find here below the PNA FISH Probe from PANAGENE:

More information on the PNA bases web page

* The PNA bases are only available in the following countries: Belgium, France, Germany, Italy, Luxembourg, Netherlands, Denmark, Ireland, United Kingdom, Greece, Portugal, Spain, Austria, Finland, Sweden, Cyprus, Czech Republic, Estonia, Hungary, Latvia, Lithuania, Malta, Poland, Slovakia, Slovenia, Bulgaria, Romania, Norway, Iceland, Bosnia, Serbia, Montenegro, Turkey, Switzerland, Israel, Russia and African countries.

The backbone of PNAs (Peptide Nucleic Acid) is made of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Despite this radical structural change, PNAs are capable of sequence-specific binding in a helix form to its complementary DNA or RNA sequence. Due to its superior binding affinity and chemical/biological stability, PNA has been widely applied in the field of biology.



PNA FISH Probe Details

  Quantity Sequence Specificity
C-rich Telomere Probe 5 nmol repeats of CCCTAA For both human and mouse
G-rich Telomere Probe 5 nmol repeats of TTAGGG For both human and mouse
Centromere Probes 5 nmol AAACTAGACAGAAGCATT For both human and mouse
Centromere Protein B Probes 5 nmol ATTCGTTGGAAACGGGA Human specific


Centromere probe

Thanks to the high sequence specifity and improved stability of PNA, chromosome aberrations in the centromere such as trisomy, polysomy and break can be clearly detected by using PNA FISH probes.

Red fluorescent probe Green fluorescent probe

Telomere probe

In principle, fluorescence in situ hybridisation (FISH) should be able to provide information on telomere length of individual chromosomes. However the efficiency of DNA/RNA oligonucleotide hybridizations for telomeric repeats investigation is not sufficient. In comparison the use of PNA stabilizes PNA-DNA/RNA duplexes. When used with PNA probes, PNA-DNA hybridization is much faster within a couple of hours and results in significantly reduced background noise due to the high signal to background noise ratio.

Telomere C probe

Red fluorescent probe Green fluorescent probe

Telomere G probe

Red fluorescent probe Green fluorescent probe

Centromeres and telomeres co-staining



Telomere and centromere co-staining Telomere and centromere co-staining Telomere and centromere co-staining
Blood lymphocytes. Centromeres and telomeres were stained with FITC-labeled PNA probe and Cy-3-labeled PNA probe, respectively. Images supplied courtesy of Radhia M’kacher, Cell Environment Genetic Toxicology section 75020 Paris, France

Protocol of PNA Telomere probe for FISH

(Download the PDF version)

PREPARATION

  • l. Preparation of the Telomere PNA probe


    • 1. Centrifuge the tubes before opening them in order to collect lyophilized PNA probe at the bottom of the tubes.
    • 2. Add formamide to the tube to obtain a stock solution of PNA probe.
    • 3. Dilute the stock solution in PNA hybridization buffer to a final concentration of 200 nM.
      ! Note: Store the PNA probe solution in the dark at 4°C.
  • ll. Preparation of solution


    • 1. Hybridization buffer
      20 mM Na2HPO4, pH 7 .4
      20 mM Tris, pH 7.4
      60% formamide
      2X SSC
      0.1 µg/ml salmon sperm DNA
    • 2. RNase A solution
    • 100 µg/ml RNase A in 2X SSC
    • 3. Pepsin 0.005% solution
    • 50 µl of 5% Pepsin stock in 50 ml 0.01 M HCl. ! Note: Make fresh ! Warm to 45 °C before use.
    • 4. Washing solution
      2X SSC/0.1% Tween-20

HYBRIDIZATION and WASHES

  • l. Pretreatment


    • 1. Prepare slide by procedure recommended for a particular cell line and air dry.
    • 2. lmmerse the slide in PBS for 15 min.
    • 3. Fix the slide in 4% formaldehyde in PBS for 4 min at 37°C.
    • 4. Wash in PBS for 5 min at 37°C (X2).
    • 5. Add 500 µl of RNase A solution to a clean plate and put each slide face down on the RNase A solution for 1 hr at 37°C.
    • ! Note: Be careful not to be dry.
    • 6. Wash in 2X SSC (X3 repeat) and wash in DW.
    • 7. lmmerse the slide in 0.005% Pepsin for 4 min at 37°C.
    • 8. Wash in PBS for 3 min at 37°C (X2).
    • 9. Repeat step 3-4.
    • 10. Wash in PBS for 5 min at room temperature.
    • 11. Dehydrate slides in cold ethanol series (for 1 min in 70%, 85%, 100%)
    • 12. Dry slide on air.
  • ll. Hybridization


    • 1. Place the slide in a pre-heated incubator at 80°C for 5 min.
    • 2. Add 20 µl of PNA probe in Hybridization buffer to the marked area on each slide.
    • ! Note: Heat Hybridization buffer for 5 min at 90'C before use,
    • 3. Cover the marked area on each slide with 18x18 mm coverslip.
      ! Note: Be careful not to be dry.
    • 4. Denature slide for 10 min at 85°C.
    • ! Note: Denaturation should be performed between at minimum 80°C and maximum 90°C.
      Check the temperature of the incubator carefully.
      Denaturing temperature below 75°C impairs result seriously.
    • 5. Place the slides in the dark at room temperature for 1hr.
  • lll. Washing


    • 1. lmmerse the slide in Washing solution at room temperature to remove the coverslips,
    • 2. Wash the slide in Washing solution for 10 min at 55-60°C (X2).
    • ! Note: Wash should be performed at 55-60°C.
    • 3. Wash the slide in Washing solution for 1min at room temperature.
  • lV. Counterstaining


    • 1. Stain the slide for 10 minutes in the DAPI/2X SSC.
    • 2. Wash the slide in 2X SSC for 2 min.
    • 3. Wash the slide in 1X SSC for 2 min.
    • 4. Wash the slide in DW for 2 min.
    • 5. Dry the slide by centrifuge.
    • 6. Add a drop of mounting media to the target area of the slide,
    • 7. Cover with a coverslip and allow the solution to spread evenly under the coverslip. Avoid air bubbles.
    • 8. Observe the stained slide using an fluorescence microscope with the appropriate filters.

References

  • 1. Kentaro Taemura et al. 2005, Developmental Biology 281, 196-207.
  • 2. Won-Woo Lee et al. 2002,Immunology 105, 458-465.
  • 3. Heather Perry. et al. 2001, Journal of Microbiological Methods 47, 281-292.
  • 4. Caifu Chen et al. 1999, Mammalian Genome 10, 13-18.
  • 5. M. Hultdin et al. 1998, Nucleic Acids Research 26(16), 3651-3656.
  • 6. Peter M. Landsdorp et al. 1996, Human Molecular Genetics 5(5), 685-691.
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