Homogeneous hybridization assay

Homogeneous hybridization assays are analyses for the detection of a given DNA/RNA sequence by hybridization without separation processes (B/F separation). These assays form a basis of real-time PCR as described below. Furthermore, sufficiently sensitive homogeneous hybridization assays could be done without PCR and enable, for example, direct detections of certain mRNA forms expressed in cells. This type of experiment has been actually performed with some organic fluorophores (Peng et al., 2005 Santangelo et al., 2006). 

Fig. 14. Examples of homogeneous hybridization assay methods (F luminophore, Q quencher, D donor, A acceptor). Thick lines represent DNA strands. Open circles on DNA strands indicate a SNP/mutation site for Molecular Beacon and insertion/deletion sites for dual FRET probe and dual FRET Molecular Beacon when these methods are applied to SNP/mutation typing or deletion/insertion detection. The solid circle on die strand indicates the complementary site.

Sueda, S., Yuan, J.L., and Matsumoto, K. (2000) Homogeneous DNA hybridization assay by using europium luminescence energy transfer. Bioconjugate Chemistry, 11, 827-831. 

Utmost importance as a criterion for the validation of a complex to be of use for the design of robust bioassays. Noteworthily this quenching effect, basically undesired, can be turned into an advantage to design homogeneous TRF assays for hybridization [26] and assay for uric acid [25]. 

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