ABSTRACT
In practice, this approach generally requires careful optimization of hybridization conditions because probe melting temperatures are very sensitive to sequence context as well as to the nature and position of the mismatched base. This difficulty in choosing optimum hybridization conditions (buffer and temperature) can be eliminated by continuous monitoring of probe hybridization as the temperature is increased, allowing the melting temperature for matched and mismatched probes to be determined directly in a process known as dynamic allele-specific hybridization (DASH; Howell, 1999 #46). Alternatively, the use of highly parallel microarrays enables an SNP-containing template to be interrogated by many different hybridization probes for each SNP [15], providing a redundancy of analysis that effectively increases the signal-to-noise ratio. Peptide nucleic acids (PNAs), which are oligonucleotide analogues with a structure that make hybridization very dependent on correct base pairing (and thus very sensitive to mismatches [16,17], can improve hybridization-based assays. Locked nucleic acids (LNAs; Wengel, 1999 #209), another oligonucleotide analogue, are likely to be similarly useful.
