title: Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models.
abstract: Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.
authors.affiliation: Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, United States of America.
authors.forename: Dawn N
authors.initials: DN
authors.lastname: Birdsell
authors.forename: Talima
authors.initials: T
authors.lastname: Pearson
authors.forename: Erin P
authors.initials: EP
authors.lastname: Price
authors.forename: Heidie M
authors.initials: HM
authors.lastname: Hornstra
authors.forename: Roxanne D
authors.initials: RD
authors.lastname: Nera
authors.forename: Nathan
authors.initials: N
authors.lastname: Stone
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Gruendike
authors.forename: Emily L
authors.initials: EL
authors.lastname: Kaufman
authors.forename: Amanda H
authors.initials: AH
authors.lastname: Pettus
authors.forename: Audriana N
authors.initials: AN
authors.lastname: Hurbon
authors.forename: Jordan L
authors.initials: JL
authors.lastname: Buchhagen
authors.forename: N Jane
authors.initials: NJ
authors.lastname: Harms
authors.forename: Gvantsa
authors.initials: G
authors.lastname: Chanturia
authors.forename: Miklos
authors.initials: M
authors.lastname: Gyuranecz
authors.forename: David M
authors.initials: DM
authors.lastname: Wagner
authors.forename: Paul S
authors.initials: PS
authors.lastname: Keim
mesh_terms: D000483:Alleles; D001419:Bacteria / Q000235:genetics* / Q000472:pathogenicity; D001431:Bacteriological Techniques / Q000191:economics; D001482:Base Composition; D020137:Base Pair Mismatch; D001483:Base Sequence; D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016680:Genome, Bacterial; D005838:Genotype; D006801:Humans; D008957:Models, Genetic; D021141:Nucleic Acid Amplification Techniques; D009691:Nucleic Acid Denaturation; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial