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Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification

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Abstract
GMO quantification, based on real-time PCR relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.
Keywords
quantification, GMO, primer/template mismatch, Genetically modified organism, maize, real-time PCR, GENETICALLY-MODIFIED MAIZE, POLYMERASE-CHAIN-REACTION, TAQ DNA-POLYMERASE, REFERENCE MOLECULES, MODIFIED ORGANISMS, MAYS L., QUANTITATION, POLYMORPHISM, DISCRIMINATION, TECHNOLOGY

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Chicago
Ghedira, Rim, Nina Papazova, Marnik Vuylsteke, Tom Ruttink, Isabel Taverniers, and Marc De Loose. 2009. “Assessment of Primer/template Mismatch Effects on Real-time PCR Amplification of Target Taxa for GMO Quantification.” Journal of Agricultural and Food Chemistry 57 (20): 9370–9377.
APA
Ghedira, R., Papazova, N., Vuylsteke, M., Ruttink, T., Taverniers, I., & De Loose, M. (2009). Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 57(20), 9370–9377.
Vancouver
1.
Ghedira R, Papazova N, Vuylsteke M, Ruttink T, Taverniers I, De Loose M. Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY. 2009;57(20):9370–7.
MLA
Ghedira, Rim, Nina Papazova, Marnik Vuylsteke, et al. “Assessment of Primer/template Mismatch Effects on Real-time PCR Amplification of Target Taxa for GMO Quantification.” JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 57.20 (2009): 9370–9377. Print.
@article{772476,
  abstract     = {GMO quantification, based on real-time PCR relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.},
  author       = {Ghedira, Rim and Papazova, Nina and Vuylsteke, Marnik and Ruttink, Tom and Taverniers, Isabel and De Loose, Marc},
  issn         = {0021-8561},
  journal      = {JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY},
  language     = {eng},
  number       = {20},
  pages        = {9370--9377},
  title        = {Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification},
  url          = {http://dx.doi.org/10.1021/jf901976a},
  volume       = {57},
  year         = {2009},
}

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