Hybridization-based assay

The ability to synthesize short oligonucleotide DNA probes with an acridinium ester attached at any defined position within the sequence (A16, N5) has allowed for the use of such probes in hybridization-based assays for RNA and DNA target sequences. Two techniques have recently emerged, together with a combination of the two (for enhanced sensitivity). 

Vlieger AM, Medenblik AM, van Gijlswijk RP, Tanke HJ, van der Ploeg M, Gratama JW, and Raap AK (1992) Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection. Analytical Biochemistry 205(1) 1-7. 

Other methods have been developed that allow the detection of point mutations in non-hybridization based assays. An effective alternative electrochemical method harnesses differences in the kinetics of the reaction of Ru(bpy)3 with gnanine in the context of base mismatches to report base substitutions [47]. In addition, altered patterns of chemical reactivity have been detected in RNA-DNA hybrids containing 2-NH2 modifications in an RNA complement at mispaired positions [48]. Extension of this approach to an immobilized system has not yet been demonstrated, but it may hold pronfise for any applications where alterations in chemical, rather than electrochanical, reactivity are required. 

The three main categories of hybridization probes for real-time PCR are (1) cleavage based assays such as TaqMan, (2) displaceable probe assays such as Molecular Beacons and (3) probes which are incorporated directly into primers such as Scorpions. 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

However, if the gene has been cloned, but the required activity is not produced, then the functional test will fail to pick up the target gene. In this case, if some gene sequence information is available, then it may be possible to test for the presence of DNA with the expected sequence by hybridization with radio-labelled probe DNA or, more usually, by PCR. This sequence-based screening test could pick up positives which have been missed in the initial screen because the gene has been successfully cloned but the enzyme has not been produced in an active form (perhaps because expression has not occurred or because E. coli is a poor host to support production of active enzyme), or where there is no convenient function-based assay available. 

Another application of this methodology is to use several different copolymers to detect multiple analytes in a single sample. Each polymer would have a different antibody attached and be precipitated at a different temperature. This appears to be possible since one copolymer that precipitates at a lower temperature does not appear to remove a copolymer with a higher LCST from solution. (J. H. Priest, unpublished observations). It is also conceivable that a DNA probe-based assay could be included with a panel of immunoassays. After removal of the various antibody-copolymer conjugates at lower temperature sodium chloride would be added, the hybridization would be performed at 55 C and the DNA-copolymer conjugates precipitated at 65 C. 

Fig. 1. A simplified flow chart for dual-color bright-field in situ hybridization (BISH) assays using horseradish peroxidase (HRP) and/or alkaline phosphatase (AP)-based immunological signal detections.

Solution-based nucleic acid hybridization assays represent a class of analytical methodologies that provide for detection of target-probe hybridization events. Solution-based assays offer several advantages in comparison to configurations that use surface-bound probes, primarily from the standpoint of negating the requirement for immobilization of a probe sequence to a solid surface. Therefore, thermodynamic, kinetic and adsorptive effects that are relevant in terms of consideration of hybridization at interfaces are not encountered. Solution-based hybridization assays provide a simple way of detecting hybridization events in real-time. 

Schutz E, von Ahsen N, Oellerich M. Genotyping of eight thiopurine methyltransferase mutations three-color multiplexing, two-color/shared anchor, and fluorescence-quenching hybridization probe assays based on thermodynamic nearest-neighbor probe design. Clin Chem 2000 46 1728-1737. 

Controversy exists as to the most useful assay for HER2-overexpres-sion, particularly for identification of candidates for trastuzumab therapy. Following the approval of trastuzumab, an IHC-based assay (Herceptest), similar but not identical to the trastuzumab clinical trials assay, was developed for this use. Fluorescence in situ hybridization (FISH)-based assays have also been developed to determine HER2 gene amplification. The concordance of these assays with each other and with the trastuzumab clinical trials assay has been questioned (252). Since the probability of response to trastuzumab therapy appears to correlate with the level of HER2 overexpression, further studies will be required to establish standardized HER2 testing procedures. 

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. 

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