Hybridisation probes

Recently, introduction of DNA analogous detection systems such as the use of peptide nucleic acid (PNA) technology has attracted considerable attention [10]. The PNAs are synthetic analogues of DNA that hybridise with complementary DNAs or RNAs with high affinity and specificity, essentially because of an uncharged and flexible polyamide backbone. The unique physico-chemical properties of PNAs have led to the development of a variety of research and diagnostic assays where these are used as molecular hybridisation probes [11]. 

R. Erhadt, D. Bradford, R. J. Sevior, R. Amann and L. L. Blackall (1997). Development and use of fluorescent in situ hybridisation probes for the detection and identification of Microtrix parvicella in activated sludge. Syst. Appl. Microbiol., 20, 310-318. 

More recently double stranded DNA-binding dyes, (e.g., SYBR Green), have been introduced (Giulietti et al. 2001) which removed the need for an expensive, specific probe to be designed. Other sophisticated tools have been developed to work in conjunction with the Taqman method, for example molecular beacons, scorpions and hybridisation probes. These techniques rely on the FRET (Fluorescence Resonance Energy Transfer) principle but do not require the nuclease activity of the Taq polymerase. The different real-time... 

DNA probes (hybridisation probes) are nsed to identify and label DNA fragments that contain a specific seqnence. A probe is simply a short length of DNA (20-100 nncleotides long) with a label attached. There are two common types nsed ... 

Analogues chelated to metal ions have been incorporated into oligonucleotides. The incorporation into an 11-mer DNA duplex of the bipyridine ligand (bpy), (78), such that two bpy units are complementary, yields a duplex that can be stabilised by copper ions. In the presence of copper, the duplex melts 10°C higher. In another report, phenanthroline-based (79) nucleosides have been incorporated into ODNs as hybridisation probes. Hairpin duplexes have been synthesised containing two phenanthroline nucleosides, one containing Ru" which exhibits a long-lived excited state, the other Os , which is non-emissive... 

The synthesis and characterisation of photocleavable peptide-DNA conjugates have been studied. The peptide portions have been used as mass tags, released during ionisation by UV-MALDI to identify unique DNA sequences. The conjugates were evaluated as hybridisation probes, and the photocleavable peptide had no effect on the thermal stability of DNA duplexes. 

Analysis of Peptide Nucleic Acid Hybridisation Probes... 

We describe two approaches to SNP analysis by MALDI-TOF MS, one involving the analysis of peptide nucleic add hybridisation probes [10], and the other the analysis of products of a novel, enzymatic invasive cleavage assay [11]. 

ANALYSIS OF PEPTIDE NUCLEIC ACID HYBRIDISATION PROBES... 

The approach using PNA hybridisation probes for MALDI-TOF mass spectrometric analysis is comprised of the following steps (Figure lb) immobilisation of biotinylated target DNA (e.g. a PCR amplicon) by binding to streptavidin coated magnetic beads dissociation and removal of the non-biotinylated... 

A number of applications have been described relying on fluorophores, and a strategy for high-throughput analysis of DNA-protein interactions has been reported/ Fluorescence has been used to monitor DNA base excision repair/ to monitor the activity of the endonuclease XPF-ERCCl/ as hybridisation probes/ for the detection of protein interactions/ in PCR and in fluorescence correlation spectroscopy... 

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