| Due to their usefulness in tracking the evolution of man and the discovery that they cause many different diseases, the scientific community has become extremely interested in mitochondrial DNA (mtDNA) mutations. PCR is a key component in nearly all genotyping methods, but using it can be problematic for mtDNA since mtDNA's highly polymorphic nature makes it difficult to design adequate primers. This is because polymorphisms can easily obscure the source sequence, preventing primers from annealing properly. For this reason, we modified a popular online primer design program, Primer3, to create mtPrimer3, a primer design program specially designed to create primers for mtDNA. mtPrimer3 creates primers that avoid highly polymorphic areas, to greatly increase the chances that the primers will properly anneal, and include areas that are unique to mtDNA only, so that false positive results are not generated by accidental annealing to nuclear DNA. This is accomplished by (1) checking the stretch of sequence that a primer anneals to against a database of known mitochondrial polymorphisms, and (2) by submitting the primers to BLAST to see if there is significant homology with human genomic DNA. If a primer pair has too many polymorphisms, then it is disqualified from being a "good" primer pair. The primer pairs that have been submitted to BLAST are scored based on a custom-designed scoring system, a harmonic mean that negatively penalizes primers with low expect values. The primer pairs are then ordered from greatest to least and the top scoring primer pairs are emailed to the user. In this way, mtPrimer3 creates primers that are suited for working with mtDNA. |
