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.

Multiple mutations in the para -sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico

“…Furthermore, we suspected this relationship might be stronger if we focused on the proportion of individuals that carried more than one resistance SNP. We We designed Melt-MAMA qPCR assays [38] for three SNP positions. Two assays distinguish susceptible versus resistance SNPs previously described in the exons for domain II C190A and domain III T2134A [25,27].…”

“…Real-time qPCR assays were developed for Rhipicephalus microplus based on the Melt-MAMA platform [38] to rapidly screen thousands of ticks for resistance SNPs at three loci in the para-sodium channel gene.…”

“…Agarose-MAMA [38] assays for Rhipicephalus microplus enable screening three resistance SNP loci in the para-sodium channel gene without the need for a real-time qPCR instrument.…”

Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype <i>Bacillus anthracis</i>

“…Based on its simplicity, low cost, non-destructive nature, high sensitivity and specificity, the popularity of HRM analysis has grown considerably in the last few years. HRM analysis is an effective alternative to other [29]. HRM is an ideal format for scoring a small number of SNPs.…”

Antimicrobial susceptibility of Bacillus anthracis strains from Hungary

“…Briefly, the strains were identified on species level with a dual-probe TaqMan assay targeting a single nucleotide polymorphism (SNP) in the plcR gene (Easterday et al, 2005). The genotypes of the isolates were determined with melt mismatch amplification mutation assays according to Birdsell et al (2012) targeting the SNPs identified by Van Ert et al (2007).…”