Failed PCR amplifications of MBP-STR alleles due to polymorphism in the primer annealing region.
The PCR-based STR system MBP-B (myelin basic protein locus B) has been reported to exhibit a high rate of mutations. Using a newly designed pair of primers we present evidence that this is due to failed amplifications caused by a polymorphism in the annealing region of the reverse primer originally designed. With the new reverse primer described here no exclusions were found (out of 59 mother/child pairs analysed) while one was detected with the old set of primers. The results obtained with both pairs of primers in a random population sample (n = 112) from 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 primer binding sequence originally described was determined. This substitution is located upstream from the repetition site and consists of G-->A transition. This variation reaches polymorphic frequency and is responsible for the relatively frequent null alleles due to failed amplifications when the previously designed primers are used.
Gusmão
L
L
Instituto de Antropologia, da Universidade do Porto, Portugal.
Amorim
A
A
Prata
M J
MJ
Pereira
L
L
Lareu
M V
MV
Carracedo
A
A
D000483:Alleles; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics*; D005260:Female; D005784:Gene Amplification / Q000235:genetics; D006580:Genetic Carrier Screening; D006720:Homozygote; D006801:Humans; D007223:Infant; D008297:Male; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics*; D010334:Paternity; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics*; D011174:Portugal; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004676:Myelin Basic Protein