The principle is to design a primer that will preferentially amplify one allele over another. Specificity of amplification can be obtained if the oligonucleotide matches the desired allele, but is mismatched to the other allele at the 3’ end of the oligonucleotide. The desired allele is readily amplified, while the mismatched allele is poorly amplified if at all. The poor amplification is a result of the mismatch between DNA template and the oligonucleotide primer that prevents efficient 3’ elongation by the polymerase. The loss of efficiency is depending on the actual mismatched pair of nucleotides, the number of mismatches (e.g., you can introduce a second mismatch 5’ to the first one) and the type of polymerase you are using. Primers were designed using the Oligo programme (version 4.1) [127].
© 2001 Alexander Binder