Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays

Abstract

Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.

Peptide nucleic acids on microarrays and other biosensors

“…Therefore, the cost of synthesis should fall if the consumption of PNA molecules were to increase. In terms of specificity, unspecific binding events, such as in cases of polypurine sequences, have been reported [18]. However, this is no different to the behavior of DNA molecules of the same sequence but is made more apparent by the increased duplex stability.…”

“…The uses of PNA as antisense probe [9], DNA opener [10] or for polymerase chain reaction (PCR) clamping [11,12] [18,19] are typical examples. Assays of this latter analytical format are reviewed below.…”

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps

“…Thus, target binding to immobilized PNA under low-salt conditions may have certain advantages not only within the context of solid-phase nucleic acid purification strategies, but also as capture-detection probes in a microarray format (56). Under high-salt extraction and purification conditions, however, there was no evidence that PNA probes (clamps or oligomers) recover any more 16S rDNA or rRNA target than a comparable DNA oligomer in any of the environmental extracts tested here.…”

SNP genotyping using microsphere‐linked PNA and flow cytometric detection

“…They are easily manufactured and modified and have good hybridization properties compared with DNA oligos. PNA has been used in a handful of SNP genotyping assays (13)(14)(15)(16)(17)(18)(19) and as RNA capture probe coupled to the surface of microspheres (25).…”