FAILED PCR AMPLIFICATIONS OF MBP-STR ALLELES DUE TO POLYMORPHISM IN THE PRIMER ANNEALING REGION

The PCR-based TR system MBP-H (myelin basic protein locus B) has been reported to exhibit a high rate of mutations. Using a newly designed p air of primers we present evidence that this is due to failed amplific ations caused by a polymorphism in the annealing region of the reverse primer originally designed. With the new reverse primer described her e no exclusions were found (out of 59 mother/child pairs analysed) whi le one was detected with the old set of primers. The results obtained with both pairs of primers in a random population sample (n = 112) fro m North Portugal are compared. In this sample 13 individuals typed as homozygotes with the pair of primers originally described, were found to be heterozygous when the amplifications were performed with the new reverse primer. By sequence analysis, a substitution in the reverse p rimer binding sequence originally described was determined. This subst itution is located upstream from the repetition site and consists of G -->A transition. This variation reaches polymorphic frequency and is r esponsible for the relatively frequent null alleles due to failed ampl ifications when the previously designed primers are used.