Melt analysis of mismatch amplification mutation assays (melt-MAMA)

A functional study of a cost-effective snp genotyping assay in bacterial models

Dawn N. Birdsell, Talima R Pearson, Erin P. Price, Heidie M. Hornstra, Roxanne D. Nera, Nathan Stone, Jeffrey Gruendike, Emily L. Kaufman, Amanda H. Pettus, Audriana N. Hurbon, Jordan L. Buchhagen, N. Jane Harms, Gvantsa Chanturia, Miklos Gyuranecz, David M Wagner, Paul S Keim

Research output: Contribution to journalArticle

50 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere32866
JournalPLoS One
Volume7
Issue number3
DOIs
StatePublished - Mar 16 2012

Fingerprint

genotyping
Single Nucleotide Polymorphism
Amplification
Assays
Costs and Cost Analysis
Mutation
Base Composition
assays
Costs
single nucleotide polymorphism
Polymorphism
Genome Size
Nucleotides
probes (equipment)
Agar Gel Electrophoresis
Cost-Benefit Analysis
Real-Time Polymerase Chain Reaction
Genotype
Genome
Technology

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

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 R; 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.

In: PLoS One, Vol. 7, No. 3, e32866, 16.03.2012.

Research output: Contribution to journalArticle

Birdsell, DN, Pearson, TR, Price, EP, Hornstra, HM, Nera, RD, Stone, N, Gruendike, J, Kaufman, EL, Pettus, AH, Hurbon, AN, Buchhagen, JL, Harms, NJ, Chanturia, G, Gyuranecz, M, Wagner, DM & Keim, PS 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, vol. 7, no. 3, e32866. https://doi.org/10.1371/journal.pone.0032866
Birdsell, Dawn N. ; Pearson, Talima R ; 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. In: PLoS One. 2012 ; Vol. 7, No. 3.
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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.",
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