Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models

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. Effi...

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Main Authors:	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.
Format:	Online
Language:	English
Published:	Public Library of Science 2012
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306377/

id	pubmed-3306377
recordtype	oai_dc
spelling	pubmed-33063772012-03-21 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. Keim, Paul S. Research Article 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. Public Library of Science 2012-03-16 /pmc/articles/PMC3306377/ /pubmed/22438886 http://dx.doi.org/10.1371/journal.pone.0032866 Text en Birdsell et al. http://creativecommons.org/licenses/by/4.0/ 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 properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
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.
spellingShingle	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. Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models
author_facet	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.
author_sort	Birdsell, Dawn N.
title	Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models
title_short	Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models
title_full	Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models
title_fullStr	Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models
title_full_unstemmed	Melt Analysis of Mismatch Amplification Mutation Assays (Melt-MAMA): A Functional Study of a Cost-Effective SNP Genotyping Assay in Bacterial Models
title_sort	melt analysis of mismatch amplification mutation assays (melt-mama): a functional study of a cost-effective snp genotyping assay in bacterial models
description	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.
publisher	Public Library of Science
publishDate	2012
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306377/
_version_	1611515065010225152

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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