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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.

Show MeSH
Competition between specific and non-specific amplification at extremely low level DNA amounts.With extremely low-level DNA amounts (<∼2 copies), stochastic, spurious, non-specific amplification could outcompete allele-specific amplification. The Bacillus anthracis melt-MAMA targeting the A.Br.003 clade [4] stochastically amplified allele-specific product and non-specific spurious products at amounts of less than a single copy (∼0.19 copies). (A and B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates at 1.15 ng and at two low level ten-fold dilution series (near-single copy and less than a single copy) in replicates of eight. The number assigned to each amplification curve (3, 8–9) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curve derivatives for the 1.15 ng and lowest template amounts are shown. This panel illustrates that genotyping accuracy was not compromised at DNA template amounts near single copy level, but spurious amplification was observed in dilution points below this level. This spurious amplification had a unique melt-profile that did not match the profile of either allele types (red arrow).
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pone-0032866-g008: Competition between specific and non-specific amplification at extremely low level DNA amounts.With extremely low-level DNA amounts (<∼2 copies), stochastic, spurious, non-specific amplification could outcompete allele-specific amplification. The Bacillus anthracis melt-MAMA targeting the A.Br.003 clade [4] stochastically amplified allele-specific product and non-specific spurious products at amounts of less than a single copy (∼0.19 copies). (A and B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates at 1.15 ng and at two low level ten-fold dilution series (near-single copy and less than a single copy) in replicates of eight. The number assigned to each amplification curve (3, 8–9) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curve derivatives for the 1.15 ng and lowest template amounts are shown. This panel illustrates that genotyping accuracy was not compromised at DNA template amounts near single copy level, but spurious amplification was observed in dilution points below this level. This spurious amplification had a unique melt-profile that did not match the profile of either allele types (red arrow).

Mentions: A concern with genotyping assays is whether they are accurate at very low template concentrations, yet to our knowledge this variable has not been extensively tested with Melt-MAMA. Therefore, we investigated the accuracy and sensitivity of Melt-MAMA against a wide range of DNA template amounts using three assays that differentiate six genetic populations of the pathogen Bacillus anthracis. Ten-fold serial dilutions of SNP polymorphic allele DNA templates (ancestral and derived, respectively), of equal DNA amounts based on TaqMan assays (Figure 6) and NanoDropTM measurements were screened on three Melt-MAMAs targeting B. anthracis lineages A.Br.003, A.Br.004, and A.Br.006 [4]. Dual Probe TaqMan assays are reported to detect DNA templates at single genomic equivalent or copy levels [4], [5], [38] and our TaqMan assay result indicates that levels less than a single copy can be detected in a fashion consistent with stochastic sampling processes (Figure 6). The A.Br.003 Melt-MAMA experiments demonstrated consistent amplification and accurate genotypes for the ancestral and the derived DNA templates at an amount of ∼115 ng–∼11.5 fg (∼1.9×107 to ∼1.9 genome copies; seven-orders of magnitude range) (Figure 7). This Melt-MAMA was one order of magnitude less sensitive than the TaqMan assay (Figures 6 & 7). The A.Br.003 Melt-MAMA displayed consistent amplification and correct genotype calls to low level DNA amounts of 11.5 fg (∼1.9 copies). Below this amount (∼1.15 fg), we observed a combination of correct allele calls, non-specific amplification, or complete amplification failures (Figure 8). For example, a single spurious amplification had a unique melt-profile that did not match the profile of either allele, indicating non-specific amplification (Figure 8D, red arrow). Without close scrutiny of this unique profile, which appears similar to an ancestral genotype profile, it is possible that this profile would be assigned an incorrect allele call. Due to a single spurious amplification observed at < ∼1 copy, and in the event that such spurious amplification remains probable at ∼11.5 fg (∼2 copies), we conservatively consider the lowest limit for this A.Br.003 Melt-MAMA to be 115 fg (∼19 copies) rather than at ∼1.9 copies. Interestingly, all negative controls (n = 34) failed to amplify by 50 cycles (Figure 8, negative controls n = 8; data not shown; negative controls n = 26).


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)

Competition between specific and non-specific amplification at extremely low level DNA amounts.With extremely low-level DNA amounts (<∼2 copies), stochastic, spurious, non-specific amplification could outcompete allele-specific amplification. The Bacillus anthracis melt-MAMA targeting the A.Br.003 clade [4] stochastically amplified allele-specific product and non-specific spurious products at amounts of less than a single copy (∼0.19 copies). (A and B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates at 1.15 ng and at two low level ten-fold dilution series (near-single copy and less than a single copy) in replicates of eight. The number assigned to each amplification curve (3, 8–9) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curve derivatives for the 1.15 ng and lowest template amounts are shown. This panel illustrates that genotyping accuracy was not compromised at DNA template amounts near single copy level, but spurious amplification was observed in dilution points below this level. This spurious amplification had a unique melt-profile that did not match the profile of either allele types (red arrow).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032866-g008: Competition between specific and non-specific amplification at extremely low level DNA amounts.With extremely low-level DNA amounts (<∼2 copies), stochastic, spurious, non-specific amplification could outcompete allele-specific amplification. The Bacillus anthracis melt-MAMA targeting the A.Br.003 clade [4] stochastically amplified allele-specific product and non-specific spurious products at amounts of less than a single copy (∼0.19 copies). (A and B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates at 1.15 ng and at two low level ten-fold dilution series (near-single copy and less than a single copy) in replicates of eight. The number assigned to each amplification curve (3, 8–9) denotes the DNA amount for the starting template. (C & D) The temperature-dissociation (melt) curve derivatives for the 1.15 ng and lowest template amounts are shown. This panel illustrates that genotyping accuracy was not compromised at DNA template amounts near single copy level, but spurious amplification was observed in dilution points below this level. This spurious amplification had a unique melt-profile that did not match the profile of either allele types (red arrow).
Mentions: A concern with genotyping assays is whether they are accurate at very low template concentrations, yet to our knowledge this variable has not been extensively tested with Melt-MAMA. Therefore, we investigated the accuracy and sensitivity of Melt-MAMA against a wide range of DNA template amounts using three assays that differentiate six genetic populations of the pathogen Bacillus anthracis. Ten-fold serial dilutions of SNP polymorphic allele DNA templates (ancestral and derived, respectively), of equal DNA amounts based on TaqMan assays (Figure 6) and NanoDropTM measurements were screened on three Melt-MAMAs targeting B. anthracis lineages A.Br.003, A.Br.004, and A.Br.006 [4]. Dual Probe TaqMan assays are reported to detect DNA templates at single genomic equivalent or copy levels [4], [5], [38] and our TaqMan assay result indicates that levels less than a single copy can be detected in a fashion consistent with stochastic sampling processes (Figure 6). The A.Br.003 Melt-MAMA experiments demonstrated consistent amplification and accurate genotypes for the ancestral and the derived DNA templates at an amount of ∼115 ng–∼11.5 fg (∼1.9×107 to ∼1.9 genome copies; seven-orders of magnitude range) (Figure 7). This Melt-MAMA was one order of magnitude less sensitive than the TaqMan assay (Figures 6 & 7). The A.Br.003 Melt-MAMA displayed consistent amplification and correct genotype calls to low level DNA amounts of 11.5 fg (∼1.9 copies). Below this amount (∼1.15 fg), we observed a combination of correct allele calls, non-specific amplification, or complete amplification failures (Figure 8). For example, a single spurious amplification had a unique melt-profile that did not match the profile of either allele, indicating non-specific amplification (Figure 8D, red arrow). Without close scrutiny of this unique profile, which appears similar to an ancestral genotype profile, it is possible that this profile would be assigned an incorrect allele call. Due to a single spurious amplification observed at < ∼1 copy, and in the event that such spurious amplification remains probable at ∼11.5 fg (∼2 copies), we conservatively consider the lowest limit for this A.Br.003 Melt-MAMA to be 115 fg (∼19 copies) rather than at ∼1.9 copies. Interestingly, all negative controls (n = 34) failed to amplify by 50 cycles (Figure 8, negative controls n = 8; data not shown; negative controls n = 26).

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