Hybridization Assays—Principles

Schultz E, Galland R, Du Bouetiez D et al (2008) A novel fluorescence-based array biosensor principle and application to DNA hybridization assays. Biosens Bioelectron 23 987-994... 

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

Following principles similar to those of immunoassays, noncompetitive hybridization assays in general demonstrate better sensitivity than competitive formats of... 

Fig. 2. Principle of the standard two-hybrid assay. A bait protein is fused to a DNA-binding domain (DBD). A library of fusion between cellular proteins and a transcriptional activation domain (ACT) is introduced together with the bait protein in a yeast strain containing appropriate reporter genes harboring a cognate binding site for the DBD. The prey-ACT fusion interacting with the bait protein will activate Pol II-mediated transcription, resulting in the production of the reporter transcript that can be selected for.

The basic idea of on-chip DNA hybridization assays is to integrate assay operations such as sequential sample loading, transportation, mixing, incubation, and detection on a single microfluidic chip. Although the microfluidics-based DNA hybridization assays share the same basic principle, the formats of these assays vary. Generally, the present-day formats of the on-chip DNA hybridization assays remain the main types directed liquid flow-based on-chip DNA hybridization assays, surface-based on-chip DNA hybridization assays, bead-based on-chip DNA hybridization assays, and the on-chip DNA hybridization assays with membrane/film-based transducer. 

Window-optimized measurement is accomplished in a dual-label hybridization with dual-label (donor-quencher) probes composed of either europium or terbium donors and fluorescent or nonfluorescent acceptors. In unhybridized free form, flexibility of the probe allows very efficient energy transfer resulting in very short-lived sensitized signal. Upon hybridization, the energy transfer is decreased and decay is longer [43]. Figure 5 shows the assay principle and the respective decays in an assay. 

This chapter outlines the chemical principles for luminescent detection of target DNA in hybridization and quantitative assays that utilize the above-mentioned chemiluminogenic and bioluminogenic reagents. 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

The major reaction principles that form the basis of the genotyping technologies available today are primer extension, ligation, and hybridization. Many of the methods for SNP genotyping rely on PCR amplification of the sequence of interest prior to allele determination. Primer design and assay optimization for multiplexed and reproducible genotyping of SNPs in large sample sets have become major bottle necks in most methods used today. 

RNase protection assays (RPA) are based on the property of RNase to digest ii RNA, but not ds RNA, and its principles and applications resemble those of SI analysis (Lynn et al., 1983 Zinn et at, 1983) (Fig. 12.3). The sequence of interest is inserted in a plasmid downstream of a bacteriophage promoter (e.g., pUC118, pT7, etc.. Table 4.4). The purified plasmid is then restricted downstream of the inserted DNA and the linearized plasmid is transcribed in the presence of a labeled rNTP precursor. The transcript should be complementary to the RNA to be studied and an excess of probe is hybridized to its target. Any RNA remaining ss is then digested by one or more RNases. The size of the RNase-resistant probe and the... 

History and Principle of Hybridization Ligand-Binding Assays... 

Current methods combine at least one of four different principles of allelic discrimination (hybridization, primer extension, ligation, or restriction) with one of four different detection techniques (chemiluminescence/ fluorescence, fluorescence polarization, resonance energy transfer, and mass spectrometry). Assay formats range from (slab)- gel electrophoresis, plates, particles, fibre arrays, and microchip arrays to semi- and homogenous assays that do not require any further sample separation or purification. 

---