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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 DN, Pearson T, 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 - PLoS ONE (2012)

Bottom Line: Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations.In this study, we identified strategies that improved the success of Melt-MAMA.We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.

View Article: PubMed Central - PubMed

Affiliation: Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, United States of America.

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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Related in: MedlinePlus

Real-time PCR amplification and dissociation (melt) curve plots.B. anthracis Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (Bacillus anthracis) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.
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pone-0032866-g003: Real-time PCR amplification and dissociation (melt) curve plots.B. anthracis Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (Bacillus anthracis) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.

Mentions: Despite the enhanced allele specificity endowed by the destabilizing mismatch design [15], our Melt-MAMAs often encountered costly design failure rates using equal primer concentrations. Accurately genotyping Melt-MAMAs succeed because the two AS-MAMA primers amplify only their respective allelic templates by out-competing the non-allelic alternate AS-MAMA primer (Figures 2 & 3). Although ∼45% of our assays behave in concordance to this prediction when at equal primer concentrations, our studies also show that equal primer stoichiometry caused ∼30% of our assays to perform poorly. Among the 185 Melt-MAMAs reported here, 72 assays displayed mild to severe cross-allele primer hybridization and resulted in poor (viewed as two melt profiles for a single DNA template; data not shown) to inaccurate allele discrimination by at least one of the AS-MAMA primers (Figure 4A). To rescue these poor performing assays, the concentration of the AS-primers were altered such that the concentration of the problematic allele-specific MAMA primer was less than the alternate “weaker performing” allele-specific MAMA primer by 2x, 3x, or 4x (See Methods; Figure 4B). The degree of reduction of the problematic AS-MAMA primer depended on the severity of the cross-reactivity. This strategy of altering primer concentrations resulted in accurate genotyping for both allelic templates (Figure 4B) in all 72 cases. Unexpectedly, we observed a pattern in which the more efficient AS-MAMA primer (67 out of 72) was the one labeled with the GC-clamp (Figure 4A). Additional validation studies across a known panel of diverse samples that represent each allele further demonstrated the fidelity of these “altered primer” assays (data not shown). Following a standard assay validation strategy that includes conditions of altered primer ratios (Figure 5), increased our total assay design success rate from 46% to 87% (Table 1). Twenty-eight other assays failed for miscellaneous reasons and could not be corrected by altering the primer ratio. This final category of SNP assays required more dramatic redesign strategies, such as primer design from the opposite strand, or abandonment of the SNP locus. However, abandonment was rare and, in our hands, Melt-MAMA success was comparable and possibly superior to the successful design rate of Dual Probe TaqMan assays, which also suffer from SNP locus design constraints [23].


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

Birdsell DN, Pearson T, 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 - PLoS ONE (2012)

Real-time PCR amplification and dissociation (melt) curve plots.B. anthracis Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (Bacillus anthracis) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306377&req=5

pone-0032866-g003: Real-time PCR amplification and dissociation (melt) curve plots.B. anthracis Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (Bacillus anthracis) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.
Mentions: Despite the enhanced allele specificity endowed by the destabilizing mismatch design [15], our Melt-MAMAs often encountered costly design failure rates using equal primer concentrations. Accurately genotyping Melt-MAMAs succeed because the two AS-MAMA primers amplify only their respective allelic templates by out-competing the non-allelic alternate AS-MAMA primer (Figures 2 & 3). Although ∼45% of our assays behave in concordance to this prediction when at equal primer concentrations, our studies also show that equal primer stoichiometry caused ∼30% of our assays to perform poorly. Among the 185 Melt-MAMAs reported here, 72 assays displayed mild to severe cross-allele primer hybridization and resulted in poor (viewed as two melt profiles for a single DNA template; data not shown) to inaccurate allele discrimination by at least one of the AS-MAMA primers (Figure 4A). To rescue these poor performing assays, the concentration of the AS-primers were altered such that the concentration of the problematic allele-specific MAMA primer was less than the alternate “weaker performing” allele-specific MAMA primer by 2x, 3x, or 4x (See Methods; Figure 4B). The degree of reduction of the problematic AS-MAMA primer depended on the severity of the cross-reactivity. This strategy of altering primer concentrations resulted in accurate genotyping for both allelic templates (Figure 4B) in all 72 cases. Unexpectedly, we observed a pattern in which the more efficient AS-MAMA primer (67 out of 72) was the one labeled with the GC-clamp (Figure 4A). Additional validation studies across a known panel of diverse samples that represent each allele further demonstrated the fidelity of these “altered primer” assays (data not shown). Following a standard assay validation strategy that includes conditions of altered primer ratios (Figure 5), increased our total assay design success rate from 46% to 87% (Table 1). Twenty-eight other assays failed for miscellaneous reasons and could not be corrected by altering the primer ratio. This final category of SNP assays required more dramatic redesign strategies, such as primer design from the opposite strand, or abandonment of the SNP locus. However, abandonment was rare and, in our hands, Melt-MAMA success was comparable and possibly superior to the successful design rate of Dual Probe TaqMan assays, which also suffer from SNP locus design constraints [23].

Bottom Line: Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations.In this study, we identified strategies that improved the success of Melt-MAMA.We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.

View Article: PubMed Central - PubMed

Affiliation: Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona, United States of America.

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.

Show MeSH
Related in: MedlinePlus