Mismatch discrimination

The efficiency of mismatch discrimination was also influenced by the concentration of allele-specific pentamers, and it is interesting that the influence of this factor varied with the mismatch position. For instance, once the mismatch occurred at the first position, it could be distinguished effectively even if the concentration of the pentamer was as high as 10 iM. But the concentration of pentamer had to be decreased to 0.5 p,M to successful distinguish a mismatch if it occurred at the fourth base of the 5 terminus. 

Forman, J.E., et al. Thermodynamics of duplex formation and mismatch discrimination on photolithographically synthesized oligonucleotide arrays. ACS Symposium Series, 1998, 682, p. 206-228. 

In most SBH experiments, probe sets are deliberately designed to improve discrimination. The use of probes shorter than 25 bases inherently enhances discrimination. In addition, SBH experiments may use probe molecules with an informative oligomer core of length n surrounded by a few degenerate (variable) bases, e.g. N j-B -N, which helps enhance discrimination [12,19,31]. Using degenerate bases to surround a fixed oligomer core minimizes the problem of end mismatch discrimination because the fixed core is internalized within the probe. 

Only a few overlapping probes were needed to sequence each site due to the high full match/mismatch discrimination observed in the experiment (an over 10-fold difference on average). 

Methyl-bpy (Mebpy) and bpy have been introduced in PNA duplexes (Entry 6, Table V) (16, 110, 111). The methyl group did not affect the stabUity of the PNA double helix (16).A10bp PNA duplex with a central pair of bpys was significantly less stable than a PNA duplex with the same sequence, but a central AT base pair. The destabilization effect of bpy on the PNA duplexes was larger than the effect exerted by the same ligand in DNA duplexes. This finding correlates with the superior mismatch discrimination of PNA over DNA. 

Until recently, a detailed understanding of mismatch discrimination by DNA polymerases has been hampered by the lack of structural information on mismatch incorporation by a polymerase. The recent structure of a Pol / mismatch ternary complex has opened the gates for computational analyses of molecular basis of fidelity discrimination. Starting with this structure detailed molecular dynamics (MD) and mixed... 

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. 

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. 

Gel pads of MAGIChips could serve as micro cuvettes, which would enable massive parallel or multiplex thermodynamical studies for any DNA duplexes, triplexes, DNA binding ligands, proteins, and so forth. Thousands of melting curves could be obtained simultaneously, which cannot be accomplished with the usual UV-VIS spectrophotometer. This fact demonstrates that all mismatch discrimination and mutation detection is based on the sohd theoretical foundation of classical DNA thermodynamics. 

Thermodynamics of Duplex Formation and Mismatch Discrimination on Photolithographically Synthesized Oligonucleotide Arrays... 

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