MUPrimer: a tool for finding allele specific PCR-primers for homologous gene sequences

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Polymerase chain reaction (PCR) amplification of specific regions of DNA has proven to be a powerful tool in molecular biology research. Synthetic short oligonucleotide DNA sequences (primers) provide specificity for the reaction by annealing to the target template DNA and providing the substrate for polymerase chain extension during the PCR. Primers are generally designed by extracting sequence matches from a single input DNA target with particular base composition, length, and position within the input DNA target. However, when the template DNA is from complex genomes or in mixtures of target DNAs, primer picking must be more sophisticated to enable discrimination of closely related sequences in the PCR. MUPrimer is a program to design allele-specific PCR primers that can discriminate among highly homologous gene sequences. The input target DNA includes of closely related sequences, making this software unique. User inputs include designating a master gene, primer positions, included and excluded regions, product length, primer T[subscript-m], and maximum T[subscript-m] difference allowed between forward and reverse primers. The software analyzes the sequences and returns primer pairs with a score that captures potential specificity of the primer based on the position of mismatches within a primer. The output is visual, displaying primer positions within the master sequence, a score for each primer, and an alignment of selected primer with the non-target sequences. MUPrimer is capable of providing PCR primers that enable allele-specific PCR amplification of specific target genes when in the presence of highly similar target sequences.