A Multiplex PCR Method of Detecting Recombinant DNAs from Five Lines of Genetically Modified Maize.

Matsuoka, Takeshi; Kuribara, Hideo; Akiyama, Hiroshi; Miura, Hirohito; Goda, Yukihiro; Kusakabe, Yuko; Isshiki, Kenji; Toyoda, Masatake; Hino, Akihiro

doi:10.3358/shokueishi.42.24

Cited by 124 publications

(112 citation statements)

References 9 publications

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“…PCR amplification of target genes from soil DNA and plasmid construction A plasmid containing A-amoA, B-amoA, nosZ and nirS gene fragments was cloned by use of overlapping PCR [24]. The primer pairs BA-F/B-R (696 bp of B-amoA gene), A-F/BA-R (689 bp of A-amoA gene), ZS-F/Z-R (313 bp of nosZ gene), and S-F/ZS-R (455 bp of nirS gene) used to clone the four fragments were designed based on the sequences of the BamoA, A-amoA, nosZ, and nirS genes, respectively.…”

Section: Dna Extraction and Purificationmentioning

confidence: 99%

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

Meng

Wang

et al. 2014

Anal Bioanal Chem

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DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification of amoA genes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and of nirS and nosZ genes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containing nirS, nosZ, amoA-AOB, and amoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC-IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k=2), of the plasmid reference material was determined to be (5.19±0.41)×10 9 copies μL −1 by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.

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Section: Dna Extraction and Purificationmentioning

confidence: 99%

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

Meng

Wang

et al. 2014

Anal Bioanal Chem

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“…Algunos diseñaron PCR múltiples para distinguir cinco líneas de maíz (Matsuoka et al, 2000), algunos otros han utilizado PCR en tiempo real logrando detectar hasta 0.68% de OGM en maíz (Vaïtilongom, Pijnenburg, Gendre & Brignon, 1999). Otros grupos estandarizaron PCR en tiempo real para detectar OGM en eventos específicos de maíz y soya (Lipp, Brodman, Pietsch, Pauwels & Anklam, 1999;Vollenhofer, Burg, Schmidt & Kroath, 1999;Zimmermann, Luthy & Pauli, 2000).…”

Section: S S N 0 1 8 8 -6 2 6unclassified

Detection of transgenes in genetically modified organisms and their subproducts

Rivera-Bustamante

Trejo-Saavedra²,

Rodríguez-Negrete³

2015

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24Detección de transgenes en Organismos Genéticamente Modificados (OGM) y sus subproductos Detection of transgenes in genetically modified organisms and their subproducts RESUMEN La ingeniería genética de plantas permite la manipulación directa del ácido desoxirribonucleico (DNA, por sus siglas en inglés), posibilitando la creación de nuevos genes (transgenes), usando elementos provenientes de varios organismos y la introducción de éstos en plantas (plantas u Organismos Genéticamente Modificados [OGM]) para conferirles nuevas características. Los principales cultivos OGM son la soya, el maíz y el algodón. México está expuesto a liberaciones no controladas de OGM. La relevancia de esta liberación aumentará cuando se trate de especies cuyo centro de origen o diversidad se encuentre en nuestro país. Por lo tanto, es necesario diseñar estrategias que permitan detectar la presencia de OGM. Este trabajo tiene como objeto desarrollar y validar metodologías para la detección de OGM y sus subproductos, utilizando la técnica de reacción en cadena de la polimerasa (PCR, por sus siglas en inglés). La estandarización incluye procesos que incrementen la reproducibilidad y el uso de reactivos fácilmente accesibles para permitir su implementación en diferentes laboratorios. ABSTRACTRecombinant DNA technologies allow the assembly of new genes (transgenes) that can be introduced into plants to confer them new characteristics. The new varieties are known as transgenic crops and are included in the widely used generic term Genetically Modified Organism (GMO). Corn, soybean and cotton are the most important transgenic crops whereas the most common traits are herbicide and insect tolerance. Mexico is exposed to unintentional releases of GMO into the environment. It will cause a problem when diversity is affected. Therefore, there is a need to develop procedures that will allow the detection or identification of transgenes in GMO as well as in GMO-related products. The objective of this study is to develop and validate some procedures to detect transgenes in GMO and GMO-derived products using PCR-based assays. Increase reproducibility and the use of accessible reagents are important part of the strategy in order to standardize and facilitate its implementation through different laboratories. INTRODUCCIÓNLa biotecnología moderna, y en particular la ingeniería genética, permite la manipulación y el acceso directo a la información contenida en el ácido desoxirribonucleico (DNA, por sus siglas en inglés), incluso posibilita la creación de nuevas combinaciones de genes. Por ello, las técnicas empleadas en ingeniería genética rompen las barreras impuestas por la incompatibilidad sexual y permiten introducir en plantas genes de nuestro interés junto con los elementos genéticos importantes para que su expresión sea adecuada. Ambos componentes (genes y elementos reguladores) pueden provenir de cualquier tipo de organismos. Por ejemplo, pueden venir de otra planta, pero también de organismos más distantes como hongos, virus, bacterias y animal...

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“…The utility of screening methods could be improved by the use of multiplex PCR reactions (see below). 30 Another weakness of these screening methods is the possibility of obtaining false positive results due to contamination from non-GMO sources naturally containing the target sequences. This is the case of nos3' and P-35S, whose presence could be due to some strains of Agrobacterium tumefaciens or the Cauliflower mosaic virus respectively.…”

Section: Broad Specificity Detection Methodsmentioning

confidence: 99%

“…95 One example of the application of multiplex PCR to GMO detection has been published by Matsuoka et al, 30 who were able to identify, in a single amplification reaction, up to 5 different maize varieties approved for commercialisation in Japan. The method uses 6 pairs of primers, one for the internal control and the other for the five transgenes, with a detection limit of 0.1% for each maize variety.…”

Section: Multiplex Assaysmentioning

confidence: 99%

Detection of Genetically Modified Organisms in Foods by DNA Amplification Techniques

García‐Cañas

Cifuentes

González

2004

Critical Reviews in Food Science and Nutrition

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A Multiplex PCR Method of Detecting Recombinant DNAs from Five Lines of Genetically Modified Maize.

Cited by 124 publications

References 9 publications

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers

Detection of transgenes in genetically modified organisms and their subproducts

Detection of Genetically Modified Organisms in Foods by DNA Amplification Techniques

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