DAVIDOW@mitwibr.bitnet (06/28/91)
Dear Netters and PCR users:
Do you know of any software packages which include 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
such that amplified DNA from targeted mutant alleles and amplified
wild type DNA now differ by the presence or absence of a restriction site,
whereas the two sequences do not normally differ by the presence or absence
of a site. This method greatly expands our ability to allelotype by diagnostic
restriction digests instead of by allele specific oligonucleotide hybridization
or other more complex methods.
I have written a simple BASIC program for the case in which the mutant is a
base substitution and deposited that program on Don Gilbert's IUBIO Archive
at ftp.bio.indiana.edu. However, the cases in which the mutant is an
addition, deletion or substitution (ex. the major cystic fibrosis mutation is a
3 bp deletion) is more complex and I would rather not have to write a program
if one is already out there (I have started writing, anyway). 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 program
must output ONLY enzyme recognition sites which allow distinction between the
mutant and wild type sequences. Therefore, it needs to look at both mutant and
wild type sequences. The useful algorithm would look at the DNA sequence in the
vicinity of at least plus or minus 20 bp around the targeted area (I picked 20
because a XcmI recognition site is 15 bp from end to end, and is the longest
recognition site I know of). The algorithm would see if any 1 bp change from
a perfect match in a PCR primer 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. A
recent review of directed mismatch PCR is in Sorscher and Huang (1991)
Diagnosis of genetic disease by primer-specified restriction map modification,
with application to cystic fibrosis and retinitis pigmentosa. Lancet
337:1115-1118. Apparently, all the referenced papers using the technique
picked enzymes and primers by hand. I posted a request similar to this on the
human genome bbd last winter and, not surprisingly, got no responses.
Thanks,
Lance Davidow Internet:Davidow@vax.wi.edu Bitnet:Davidow@mitwibr
FAX(617) 891-5062