Peptide Nucleic Acids: Robust Probe Hybridization Technology

tags Peptide nucleic acid (PNA ) is an artificially synthesized polymer that is capable of binding DNA and RNA in a sequence-specific manner. Since the discovery of its distinctive binding properties, PNA has been employed in a wide variety of biomedical applications, like genetic research, diagnostics, and experimental therapeutics (1). This post will concentrate on the diagnostic PNA assays that have gained widespread use in the pathology setting and briefly touch upon other promising applications of this technologies.

Unlike DNA and RNA , which have backbones of repeating sugarphosphate units, the PNA molecule is constructed upon a pseudo-peptide backbone of N-(two-aminoethyglycine) units linked by peptide bonds, to which purine and pyrimidine bases (the distinct base-pairing units of nucleic acids) are linked by way of methylene carbonyl bonds.

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 of binding to complementary DNA and RNA sequences, but the uncharged PNA backbone confers a essential advantage to PNA probes. By eliminating the repulsive electrostatic force among classic nucleic acid probes and their complementary target strands, the neutral PNA backbone confers elevated probe affinity and thermal stability to the probe-target duplex.

Specificity of probe binding is a essential aspect of probe assay design, and the physico-chemical properties of PNA probes supply important benefits for controlling assay specificity. In assays that use conventional DNA or RNA probes, the selectivity conferred by hydrogen bonding between the complementary base pairs on the probe and target strands is offset by the repulsive ionic forces amongst the strands negatively charged backbones. Optimization of assay specificity demands a delicate balance amongst parameters such as hybridization temperature, probe concentration, length, and G-C content, and the concentrations of organic solvents and ions, generating the design of a robust assay difficult even for knowledgeable diagnosticians. The larger binding energies of PNA probe-target duplexes contributed by the uncharged PNA backbone offer several practical positive aspects for diagnostic probe assay development. The higher melting temperatures of PNA -DNA duplexes permit PNA probes to invade and overcome several problematic secondary structures in target sequences, and permit extremely stringent hybridization and wash situations to be used to improve binding specificity. The higher binding affinities of PNA probes also permit shorter probe sequences and lower probe concentrations to be employed in assays, lowering costs and decreasing possible non-distinct interactions with assay substrates and biological sample elements. Mismatches in PNA-DNA duplexes are much more de-stabilizing than in corresponding DNA -DNA duplexes, a characteristic which permits PNA s probes to distinguish single base sequence discrepancies such as point mutations and single nucleotide polymorphisms with greater selectivity than DNA or RNA probes.

One more clear benefit of PNA probe chemistry is its exceptional stability. PNA molecules are extremely resistant to each nuclease and protease enzymes, and are steady over a wider pH range than DNA or RNA molecules. Probe stability is specifically critical in diagnostic settings with potentially higher amounts of contaminating enzymes, such as assays of minimally processed biological specimens or point-of-use field applications. PNA s stability can also be employed to benefit in the design and style of simplified, fast diagnostic tests which incorporate PNA probes with other assay elements, such as sample preparation reagents, in order to consolidate and decrease actions in the assay procedure.

The majority of the industrial PNA probe products available right now are made for fluorescent in situ hybridization (FISH) assays.
Dako was an early pioneer in the improvement of PNA -primarily based tests, and in keeping with its pathology concentrate is utilizing PNA s to allow novel cancer diagnostics. The first PNA probe diagnostic products on the industry have been Telomere PNA FISH Kit.

These assays, initially conceived and created by Peter Lansdorps group at the Terry Fox laboratory of the British Columbia Cancer Reseach Center, use PNA probes to rapidly and quantitatively visualize the lengths of the telomeric repeat sequences at the ends of every single chromosome (two). The kits can be utilized to assess telomeres in humans and other vertebrate species using interphase nuclei, metaphase spreads, or flow cytometry preparations. Telomere length has been implicated as a vital regulator of a cells capacity for division, and the PNA telomere assays have confirmed to be useful tools for studying the relationship among telomere length and cancer, senescence, and other events that influence genetic longevity.

Much more lately, PNA s have been incorporated into a line of cancer cytogenetic FISH probes, exactly where they are used to boost assay overall performance. Every of the FISH products, which consist of both the Split Signal and Sub-Deletion Signal categories of FISH probes, consists of two DNA fragments (labeled with green and red fluorophores, respectively) complementary to adjacent chromosomal regions that are susceptible to re-arrangement in hematological cancers.


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