Theoretical Considerations of Hybridisation

The principle underlying the use of oligonucleotides and DNA microchips is the distinction between perfect and mismatched duplexes. The effectiveness of distinction depends on many parameters, such as the length of the probe, the position of the mismatch in the probe, the AT content, and the conditions of the hybridisation reaction [19, 20]. Because of the aforementioned parameters, centrally located mismatches are easier to locate than terminal ones, and shorter probes allow for easier match/mismatch discrimination. The only drawback of using shorter probes is that overall duplex stability decreases as the length of the ohgomer deaeases, a phenomenon that is suspected to lead to low hybridisation signals with shorter probes. 

Reaction conditions can be optimised to improve the discrimination of match/mismatch duplexes. Raising the hybridisation temperature to that of the melting temperature (T ) will enhance the discrimination between match/mismatch duplexes but decrease hybridisation [22], 

The previously mentioned differences in the stability of the matched/mismatched duplexes are valid only under equilibrium hybridisation conditions. Kinetic differences can also achieve match/mismatch distinction. Post-hybridisation washes can drastically reduce the mismatched signals without affecting the matched duplexes because of faster dissociation of mismatched duplexes. 

In the work of Khrapko et al. [7], experimental interpretations showed that if the oligonucleotide probes are immobilised in three-dimensional gel elements, the apparent dissociation temperature is, in fact, dependent on the concentration of the immobilised oligonucleotide probes. This observation was used to derive an algorithm that allows for the normalisation of oligonucleotide matrices in which a higher concentration of AT-rich and a lower concentration of GC-rich immobilised nucleotides can be used to equalise apparent dissociation temperatures of duplexes that differ in their AT content, leading to true match/mismatch discrimination. 

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