PNA probe

Stender H., Sage A., Oliveira K., Broomer a. j., Young B., Coull J. Combination of ATP-bioluminescence and PNA probes allows rapid total counts and identification of specific microorganisms in mixed populations. /. Microbiol. Methods 2001 46 69-75. 

Dose C, Seitz O (2008) Single nucleotide specific detection of DNA by native chemical ligation of fluorescence labeled PNA-probes. Bioorg Med Chem 16 65-77... 

Scheme 4 Illustration of S1 nuclease mediated digestion of the duplex of long ssDNA target and the PNA probe, and subsequent FRET between the intact regions bound to the PNA probe...

Detection of the bound PNA is possible via on-column fluorescence detection. Efficient single base-pair discrimination was achieved routinely using that technique, as shown in Fig. 7. The system was optimized to observed a difference of 25°C between the perfect match and single-mismatched PNA probe. The assay should be applicable for clinical research and diagnostics and may be automated as well. 

Fig. 6 Free-solution CE separation of PNA/DNA hybrid from excess PNA probe. M13 mpl8 ssDNA 4.2 X 10-8 M, and PNA probe 1.3 X 10-7 M. Detection LIF 488/520 nm. Buffer TBE, 7 M urea (pH 8.0). CE conditions 50-mm-i.d. polyacrylamide-coated capillary (27 cm in length and 20 cm to detector), 10 s gravity injection, separation voltage — 10 kV. Laser-induced fluorescence detection with excitation at 488 nm and emission at 520 nm. The buffer contained Triszborate (pH 8.0) with 7 M urea buffer. (From Ref. 37.)...

Fig. 7 Fluorescence intensity of the PNA/DNA hybrid vs. separation temperature. Fluorescein-labeled PNA probes with complementary 3 sequence. Voltage 20 kV. Detection LIF 488/520 nm. Buffer IX TBE/30% formamide (pH 8.3). The following M13 probes were used 5 -fluorescein-00-TTT TCC CAG TCA CGA (perfect match), 5 -fluorescein-OO-TTT TCC CAG GCA CGA (single mismatch), 5 -fluo-rescein-OO-TTT TCA CAG GCA CGA (double mismatch). (From Ref. 37.)...

Figure 10.19 Schematic representation of the operational principle of the DNA sensor based on conformational flexibility change in the PNA probe structure stimulated by hybridization, (a) before hybridization, electron transfer between Fc and electrode is possible and (b) after hybridization, formation of the duplex rigidifies the probe structure, preventing efficient electron transfer. Reproduced by permission from Ref. 140 of The Royal Society of Chemistry.

Figure 4. Staining with CEN-18 FITC-labeled PNA probe (green signals). Arrows illustrate the localization preference close to the membrane of gene-poor DNA domains.

Figure 5. Over-digested (8-minute enzymatic digestion) FFPE breast tissue stained with gene probe (red), a centromere reference PNA probe (green) and DAPI (blue). The image shows doughnut formation and uneven DNA counter stain (blue). The arrow points to a nuclei with loss of red signal. The tissue specimen has a normal 1 1 gene to reference ratio at its optimal digestion time (3-minute enzymatic digestion — data not shown).

Texas Red labeled dna FITO labeled FISH PNA probe A Mount and 1 read slides... 

The most common usage for PNA molecules are as probes of complementary nucleic acid sequences. As with other nucleic acid probes, the sequences of bases on PNA probes dictate the specificity... 

Another clear benefit of PNA probe chemistry is its exceptional stability. PNA molecules are highly resistant to both nuclease and protease enzymes, and are stable over a wider pH range than DNA or RNA molecules. Probe stability is especially important in diagnostic settings with potentially high amounts of contaminating enzymes, such as assays of minimally processed biological specimens or... 

The majority of the commercial PNA probe products available today are designed for fluorescent in situ hybridization (FISH) assays. Dako was an early pioneer in the development of PNA-based tests, and in keeping with its pathology focus is using PNAs to enable novel cancer diagnostics. The first PNA probe diagnostic products on the market were Telomere PNA FISH Kit. 

In Situ Hybridization An assay for nucleic acids on site in fixed tissue sections by the use of heat to first denature and then to reanneal with specific DNA, RNA or PNA probes. 

Fig. 14. Concentration titrations of T15 (A), T45 (B), and T75 (C) target oligonucleotides on PNA probe surfaces at 10 mM or 1 M Na+ concentration, respectively. On the right side, schematic drawings represent the overlapping of the electrostatic fields between neighboring DNA strands.

Robertson KL, Yu L, Armitage BA, Lopez AJ, Peteanu LA. Fluorescent PNA probes as hybridization labels for biological RNA. Biochemistry 2006 45 6066-6074. 

PNA probes have been used for FISH experiments, and commercial kits are available for microbial targets (17). Typically, the probes present in these kits hybridize to the abundant ribosomal RNA from bacteria. A recent paper illustrated the value... 

A third application for fluorescent PNA probes is to introduce fluorescent labels into the RNA site specifically (20). For example, PNA probes have been hybridized at exonic sites that flank consecutive splice sites in a pre-mRNA from yeast. Fdrster resonance energy transfer (FRET) donor and acceptor dyes were attached covalently to the PNAs, and low FRET efficiencies were observed when the PNAs were hybridized to the pre-mRNA. However, when hybridized to the mRNA produced by splicing, large increases in ERET have been observed both in bulk solution and on a glass slide where single-molecule measurements could be made. As in the EISH applications, the ability to use short PNA probes to deliver the fluorescent dye to a desired location decreased the likelihood that the PNA wiU disrupt the structure and the function of the RNA that is under investigation. 

An interesting new approach to DNA detection involves probes attached to polymer microspheres loaded with a scintil-lant (25). The hybridization of a complementary, radiolabeled target DNA leads to strong signal amplification because of the close proximity of the scintillant and the radiolabel. Another appealing aspect of this method is that the PNA probe can be synthesized directly on the scintillant-loaded microspheres. 

Marin VL, Armitage BA. RNA guanine quadruplex invasion by complementary and homologous PNA probes. J. Am. Chem. Soc. 2005 127 8032-8033. 

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