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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

Birdsell, Dawn N., Pearson, Talima, Price, Erin P., Hornstra, Heidie M., Nera, Roxanne D., Stone, Nathan, Gruendike, Jeffrey, Kaufman, Emily L., Pettus, Amanda H., Hurbon, Audriana N., Buchhagen, Jordan L., Harms, N. Jane, Chanturia, Gvantsa, Gyuranecz, Miklos, Wagner, David M. and Keim, Paul S. (2012). Melt analysis of mismatch amplification mutation assays (melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models. PLoS One,7(3):e32866-1-e32866-18.

Document type: Journal Article
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IRMA ID 75039815xPUB517
Title Melt analysis of mismatch amplification mutation assays (melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models
Author Birdsell, Dawn N.
Pearson, Talima
Price, Erin P.
Hornstra, Heidie M.
Nera, Roxanne D.
Stone, Nathan
Gruendike, Jeffrey
Kaufman, Emily L.
Pettus, Amanda H.
Hurbon, Audriana N.
Buchhagen, Jordan L.
Harms, N. Jane
Chanturia, Gvantsa
Gyuranecz, Miklos
Wagner, David M.
Keim, Paul S.
Journal Name PLoS One
Publication Date 2012
Volume Number 7
Issue Number 3
ISSN 1932-6203   (check CDU catalogue open catalogue search in new window)
Scopus ID 2-s2.0-84858397328
Start Page e32866-1
End Page e32866-18
Total Pages 18
Place of Publication United States
Publisher Public Library of Science
HERDC Category C1 - Journal Article (DIISR)
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.
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