DAVIDOW@mitwibr.bitnet (11/29/90)
Do you know of any computer programs for finding designed mismatch PCR primers to generate diagnostic restriction fragments in allelotyping? One of the first papers using this PCR technique is Haliassos et al. Modification of enzymatically amplified DNA for the detection of point mutations. Nuc. Acids Research (1989) 17:3606. The idea is to put a mismatch in the PCR primer near the 3' end such that amplified DNA from targeted mutant alleles and amplified wild type DNA differ by the presence or absence of a restriction site. This greatly expands our ability to allelotype by diagnostic restriction digests instead of by allele specific oligonucleotide hybridization or other more complex methods. The algorithm needed is different from that used to design possible new restriction sites in a synthetic gene for a known peptide by using alternate codons. The useful algorithm would look at the DNA sequence in the vicinity of plus or minus 13 bp around the targeted point mutation (I picked 13 because a SfiI recognition site is 13 bp from end to end and is the longest recognition site I know of). The algorithm would see if any 1 bp change created a restriction site in the mutant sequence but not in the wild type. The user could then design a PCR primer incorporating that mismatch for use in an allele detection test. A primer that created a site in the wild type but not the mutant would also be useful. However, it would not allow unambiguous diagnosis of the targeted allele since other changes (possibly polymorphic, and not even mutant) in the restriction enzyme recognition site would give the same result as the targeted mutation.