Melt analysis of mismatch amplification mutation assays (melt-MAMA): A functional study of a cost-effective snp genotyping assay in bacterial models

D N Birdsell; T Pearson; Erin P Price; H M Hornstra; R D Nera; N Stone; J Gruendike; E L Kaufman; A H Pettus; A N Hurbon; J L Buchhagen; N J Harms; G Chanturia; M Gyuranecz; D M Wagner; P S Keim

doi:10.1371/journal.pone.0032866

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Melt analysis of mismatch amplification mutation assays (melt-MAMA): A functional study of a cost-effective snp genotyping assay in bacterial models

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Melt analysis of mismatch amplification mutation assays (melt-MAMA): A functional study of a cost-effective snp genotyping assay in bacterial models

D N Birdsell, T Pearson, Erin P Price, H M Hornstra, R D Nera, N Stone, J Gruendike, E L Kaufman, A H Pettus, A N Hurbon, …

PLoS One, Vol.7(3), e32866

2012

DOI: https://doi.org/10.1371/journal.pone.0032866

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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. © 2012 Birdsell et al.

Details

Title: Melt analysis of mismatch amplification mutation assays (melt-MAMA): A functional study of a cost-effective snp genotyping assay in bacterial models
Authors: D N Birdsell (Author) - Northern Arizona University, United States
T Pearson (Author) - Northern Arizona University, United States
Erin P Price (Author) - Northern Arizona University, United States
H M Hornstra (Author) - Northern Arizona University, United States
R D Nera (Author) - Northern Arizona University, United States
N Stone (Author) - Northern Arizona University, United States
J Gruendike (Author) - Northern Arizona University, United States
E L Kaufman (Author) - Northern Arizona University, United States
A H Pettus (Author) - Northern Arizona University, United States
A N Hurbon (Author) - Northern Arizona University, United States
J L Buchhagen (Author) - Northern Arizona University, United States
N J Harms (Author) - University of Saskatchewan, Canada
G Chanturia (Author) - Northern Arizona University, United States
M Gyuranecz (Author) - Ilia State University, United States
D M Wagner (Author) - Northern Arizona University, United States
P S Keim (Author) - Northern Arizona University, United States
Publication details: PLoS One, Vol.7(3), e32866
Publisher: Public Library of Science
Date published: 2012
DOI: 10.1371/journal.pone.0032866
ISSN: 1932-6203
Copyright note: Copyright © 2012 Birdsell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Organisation Unit: University of the Sunshine Coast, Queensland; Centre for Bioinnovation
Language: English
Record Identifier: 99450554702621
Output Type: Journal article

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