Nous tenons à remercier Alan Tasker (USDA-APHIS, Riverdale, MD), Stephen Compton (Clemson), Sherry Aultman (Clemson), Craig Ramsey (USDA-APHIS, Fort Collins, CO), et Betty Marose (UMD) de l&#39;aide pour obtenir des échantillons . Nous remercions les étudiants Andrew Adrian (UA-Huntsville) et Derek Thacker (UA-Huntsville) pour leur aide dans l&#39;essai de ce protocole, et Joseph Herdy pour son travail dans le tournage de la vidéo. Ce travail a été financé par National Fish and Wildlife Foundation.

<ol>
	<li>Holm, L. G., Pancho, J. V., Herberger, J. P., Plucknett, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Geographical Atlas of World Weeds. , (1979).</li><li>CABI, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crop+Protection+Compendium.">Crop Protection Compendium.</a> Commonwealth Agricultural Bureau International (CABI). , (2007).</li><li>MacDonald, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cogongrass+(Imperata+cylindrica)+-+Biology,+ecology,+and+management.">Cogongrass (Imperata cylindrica) - Biology, ecology, and management.</a> Critical Reviews in Plant Sciences. 23, 367-380 (2004).</li><li>Talley, S. M., Cseke, L. J., Zink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+diagnostic+technologies+for+invasive+plants.">Molecular diagnostic technologies for invasive plants.</a> CPHST Fort Collins Laboratory 2009 Annual Report. , 27-28 (2009).</li><li>Dozier, H., Gaffney, J. F., McDonald, S. -. K., Johnson, E. R. R. L., Shilling, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cogongrass+in+the+United+States:+History,+ecology,+impacts,+and+management.">Cogongrass in the United States: History, ecology, impacts, and management.</a> Weed Technology. 12, 737-743 (1998).</li><li>Chikoye, D., Ekeleme, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weed+flora+and+soil+seedbanks+in+fields+dominated+by+Imperata+cylindrica+in+the+moist+savannah+of+West+Africa.">Weed flora and soil seedbanks in fields dominated by Imperata cylindrica in the moist savannah of West Africa.</a> Weed Research. 41, 475-490 (2001).</li><li>Weber, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Invasive Plant Species of the World: A Reference Guide to Environmental Weeds. , (2003).</li><li>Patterson, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shading+effects+on+growth+and+partitioning+of+plant+biomass+in+Cogongrass+(Imperata+cylindrica)+from+shaded+and+exposed+habitats.">Shading effects on growth and partitioning of plant biomass in Cogongrass (Imperata cylindrica) from shaded and exposed habitats.</a> Weed Science. 28, 735-740 (1980).</li><li>Cole, J. T., Cole, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ornamental+grass+growth+response+to+three+shade+intensities.">Ornamental grass growth response to three shade intensities.</a> Journal of Environmental Horticulture. 18, 18-22 (2000).</li><li>Bryson, C. T., Koger, C. H., Byrd, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+temperature+and+exposure+period+to+heat+on+cogongrass+(Imperata+cylindrica)+viability.">Effects of temperature and exposure period to heat on cogongrass (Imperata cylindrica) viability.</a> Weed Technology. 21, 141-144 (2007).</li><li>Capo-chichi, L. J. A., Faircloth, W. H., Williamson, A. G., Patterson, M. G., Miller, J. H., van Santen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+Dynamics+and+Genotypic+Diversity+of+Cogongrass+(Imperata+cylindrica)+at+the+Point+of+Introduction+in+the+Southeastern+United+States.">Invasion Dynamics and Genotypic Diversity of Cogongrass (Imperata cylindrica) at the Point of Introduction in the Southeastern United States.</a> Invasive Plant Science and Management. 1 (2), 133-141 (2008).</li><li>Talley, S. M., Ramsey, C. L., Zink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimentally+assessing+the+invasive+potential+of+plants.">Experimentally assessing the invasive potential of plants.</a> CPHST Laboratory 2009 Annual Report. , 29-30 (2009).</li><li>Hodkinson, T. R., Chase, M. W., Lled&#243;, M. D., Salamin, N., Renvoize, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetics+of+Miscanthus,+Saccharum+and+related+genera+(Saccharinae,+Andropogoneae,+Poaceae)+based+on+DNA+sequences+from+ITS+nuclear+ribosomal+DNA+and+plastid+trnLintron+and+trnL-F+intergenic+spacers.">Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnLintron and trnL-F intergenic spacers.</a> J. Plant Res. 115, 381-392 (2002).</li><li>Kress, W. J., Wurdack, K. J., Zimmer, E. A., Weigt, L. A., Janzen, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+DNA+barcodes+to+identify+flowering+plants.">Use of DNA barcodes to identify flowering plants.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (23), 8369-8374 (2005).</li><li>Roodt-Wilding, R., Spies, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+relationships+in+southern+African+chloridoid+grasses+(Poaceae)+based+on+nuclear+and+chloroplast+sequence+data.">Phylogenetic relationships in southern African chloridoid grasses (Poaceae) based on nuclear and chloroplast sequence data.</a> Systematics and Biodiversity. 4, 401-415 (2006).</li><li>Brody, J. R., Kern, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+boric+acid:+a+Tris-free,+cooler+conductive+medium+for+DNA+electrophoresis.">Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.</a> BioTechniques. 36, 214-216 (2004).</li><li>Chou, C. -. H., Tsai, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+the+intergenic+spacer+of+ribosomal+DNA+of+Imperata+cylindrica+(L.)+Beauv.+var.+major+(Cogongrass)+populations+in+Taiwan.">Genetic variation in the intergenic spacer of ribosomal DNA of Imperata cylindrica (L.) Beauv. var. major (Cogongrass) populations in Taiwan.</a> Botanical Bulletin of Academia Sinica (Taipei). 40, 319-332 (1999).</li></ol>

Nous n&#39;avons rien à communiquer.

<html> La pépinière des États-Unis et de l&#39;aménagement paysager se développent sur la culture et la vente d&#39;espèces de plantes exotiques et roman. Ceci, couplé avec la mondialisation croissante du commerce, augmente les chances que des espèces végétales envahissantes entreront, établir et diffuser aux Etats-Unis La capacité de réguler le gouvernement fédéral ces plantes repose sur l&#39;information qui n&#39;est souvent pas disponible, y compris le potentiel de devenir envahissantes , la taxonomie correcte, et la distinction génétique de taxons indigènes et naturalisées. Parce que notre connaissance des plantes envahissantes est souvent limitée, avec des plantes importées cachés caractéristiques envahissantes ont été introduites volontairement pour apprendre plus tard qu&#39;ils envahissent nos ressources agricoles et naturelles. Ce protocole vise à résoudre ces problèmes liés à I. variétés cylindrica en fournissant la première méthode simplifiée moléculaire qui peut distinguer précisément de type sauvage imperata cylindrique et la forme est revenue de son homologue JBG ornementales. jove_content &quot;&gt; Pour le développement de ce protocole, de type sauvage imperata cylindrique ont été recueillies auprès des populations naturalisées à l&#39;unité de l&#39;étang du ruisseau forestier à Santa Rosa County, près de Jay, Floride en Juin 2008. JBG a été obtenue à partir d&#39;une pépinière commerciale (Bluebird Nursery, Inc ..) en Juin 2008 ainsi que de la collection d&#39;un particulier de à Columbia, MO JBG revient ont été obtenus à partir de la cour de la Campbell Musée géologique à l&#39;Université de Clemson, Caroline du Sud en Juin 2008; de l&#39;Université Park à Riverdale, MA en Juin 2009, et de la cour avant d&#39;un propriétaire à Columbia, MO en 2009 (identifié par Leland Cseke). Toutes les plantes ont été maintenus dans une serre située à l&#39;Université d&#39;Alabama à Huntsville (Huntsville, AL). Le séquençage génétique de l&#39;ADN recueilli à partir de ces plantes incluses dans les comparaisons de la profondeur de 9 régions d&#39;ADN indépendantes couramment utilisées pour 2 plantes de codes à barres. Dans tous les cas, les séquences de JBG étaient un bonus de 100% à ceux de la JBG revenir, contribuant ainsi àvérifier que JBG en effet revenir à un vert, forme invasive. Seule la SA nucléaire et les chloroplastes trnL-F régions présentent des différences qui peuvent être utilisés pour distinguer génétiquement imperata cylindrique et JBG. La région ITS a un total de 3 SNP (polymorphismes nucléotidiques simples) entre imperata cylindrique et JBG, tandis que la région trnL-F dispose de 2 SNP et 2 indels (insertions et des suppressions). Ces différences génétiques a permis diverses amorces spécifiques de PCR pour être mis au point qui permet de distinguer entre de type sauvage et imperata cylindrique JBG revient. Les résultats les plus fiables proviennent de amorces dérivées de la plaste trnL-F région. Ainsi, ce protocole est basé sur les différences de séquences entre les régions trnL-F du génome chloroplastique de imperata cylindrique, JBG JBG et revenues (figure 4). Pour aider à générer des amorces qui sont plus spécifiques aux variétés en question et d&#39;aider à éviter les faux positifs à partir d&#39;espèces étroitement apparentées, all connus trnL-F séquences de I. cylindrica variétés ont été comparées avec trnL-F séquences provenant d&#39;espèces de graminées connexes (43 séquences indépendantes de 29 espèces, par exemple, Cymbopogon citratus, Sorghastrum incompletum, les Coix lacryma-jobi, de Miscanthus sinensis, de Saccharum officinarum, Sorghum halepense). Bien, nous avons examiné les séquences des amorces spécifiques à travers divers-29 espèces d&#39;herbe, la spécificité des amorces spécifiques variété n&#39;a pas été complètement examinée pour sa capacité à amplifier l&#39;ADN de la plupart des autres espèces de graminées. Par conséquent, le tissu utilisé pour l&#39;extraction d&#39;ADN doivent être soigneusement identifiés comme I. cylindrica avant de commencer ce protocole. Si l&#39;herbe ne peut pas être identifié soit comme imperata cylindrique ou JBG, nous suggérons le séquençage du produit PCR afin de s&#39;assurer que les séquences sont une correspondance exacte ou JBG imperata cylindrique. L&#39;heure actuelle, la méthode la plus exacte pour vérifier l&#39;identité d&#39;un échantillon d&#39;herbe donnée consiste à effectuer la PCR à la fois sur le trnl-F et de ses régions, suivie d&#39;une vérification séquence de produits de PCR et la comparaison des séquences avec des séquences connues de taxons identifiés avec précision. L&#39;ADN peut être amplifié en utilisant des amorces de contrôle décrites dans le présent protocole (pour la région trnL-F) ou des amorces d&#39;autres disponibles dans d&#39;autres publications 13-15. Le séquençage est beaucoup plus de travail et coûteux que d&#39;utiliser notre procédure simplifiée. La qualité des amorces utilisées pour la PCR est essentielle à la réussite de la procédure. Nous avons fait des amorces pour cette procédure disponible à partir Oblique Bio, Inc ( <a href="http://www.obliquebio.com/web/" target="_blank">http://www.obliquebio.com/web/</a> , Huntsville, AL). L&#39;avantage de commander les amorces de Bio Oblique, c&#39;est qu&#39;ils stocker un grand nombre de parties aliquotes de chaque amorce de la production en série d&#39;échantillon identique à celle des amorces que nous avons utilisées pour l&#39;optimisation dans notre laboratoire. Par conséquent, les amorces non seulement avoir la même séquence, mais they proviennent du même lot de production exacte qui a été utilisé dans ce protocole. En utilisant des amorces provenant du même lot, peut aider à éviter les variables parasites dans la procédure qui pourrait résulter de différences dans la qualité des amorces de PCR. De même, tandis que d&#39;autres polymérases Taq devrait fonctionner correctement pour la PCR, la qualité de la Taq polymérase utilisée aura un impact sur la qualité des résultats de PCR. Afin de permettre une meilleure cohérence dans les réactifs de PCR, nous avons optimisé le protocole en utilisant des réactifs de Clontech. Le Avantage 2 polymérase (Clontech, Mountain View, CA, Cat # 639201 ou 639202) est un mélange d&#39;un solide, démarrage à chaud Taq polymérase et une enzyme de relecture qui permet de fournir une haute spécificité et plus précises amplifications. Parce que ce protocole repose sur l&#39;ADN des chloroplastes, qui est hérité de la mère dans les herbes, les événements d&#39;hybridation entre imperata cylindrique et les génotypes JBG ne peut pas être capturé avec notre procédure d&#39;identification moléculaire. Dans les cas où hypridization est suspeDECT, nous recommandons d&#39;utiliser les régions nucléaires qui sont héritées des deux parents. Le plus couramment utilisé non-plaste région variable à considérer dans le génotypage des plantes est le ribosomique nucléaire région ITS 13-15,17. Actuellement, nous faisons des progrès vers le multiplexage de l&#39;amplification du chloroplaste trnL-F région avec celle de la centrale nucléaire de sa région dans le même tube PCR. Multiplexage plaste avec les régions d&#39;ADN nucléaire serait potentiellement contourner les limites de l&#39;utilisation soit seul, mais de telles méthodes nécessitent l&#39;optimisation et l&#39;évaluation supplémentaire pour déterminer la faisabilité au cas par cas. L&#39;utilisation d&#39;indicateurs quantitatifs PCR en temps réel (qPCR) et les nouvelles technologies, telles que les balises moléculaires (sondes fluorescentes d&#39;amorces), sont également en cours d&#39;évaluation à sécurité intrinsèque et précise les méthodes de génotypage des plantes. Le protocole présenté ici fournit une approche rapide et fiable de distinguer le JBG revenir de celle de type sauvage imperata cylindrique. Nous encourageons l&#39;utilisationrs de ce protocole à nous contacter pour signaler les résultats découlant de l&#39;utilisation de ce protocole. Cette information partagée permettra de fournir des informations sur la distribution de JBG revient. Cela permettra également d&#39;aider les régulateurs à l&#39;USDA de prendre des décisions éclairées sur les mesures qui peuvent être nécessaires pour contourner la propagation et l&#39;hybridation potentiel des variétés imperata cylindrique hautement invasifs.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> DNeasy usine Mini Kit </td><td> Qiagen, Valencia, CA </td><td> 69104 ou 69106 </td><td> Toute kit génomique réputé plante à ADN qui donne l&#39;ADN de bonne qualité devrait fonctionner correctement pour ces procédures. </td></tr><tr><td> trnF (GAA)-F </td><td> Oblique Bio, Inc </td><td> 3-0578 </td><td> Amorce le contrôle positif avant </td></tr><tr><td> trnL (exon 5 &#39;)-C </td><td> Oblique Bio, Inc </td><td> 3-0579 </td><td> Inverser amorce contrôle positif </td></tr><tr><td> trnLF-C-F1 </td><td> Oblique Bio, Inc </td><td> 3-0864 </td><td> Transférer de type sauvage amorce imperata cylindrique </td></tr><tr><td> trnLF-C-R1 </td><td> Oblique Bio, Inc </td><td> 3-0865 </td><td> Inverserde type sauvage amorce imperata cylindrique </td></tr><tr><td> trnLF-R-F2 </td><td> Oblique Bio, Inc </td><td> 3-0866 </td><td> Transférer JBG JBG et revenir amorce </td></tr><tr><td> trnLF-R-R2 </td><td> Oblique Bio, Inc </td><td> 3-0867 </td><td> Inverser JBG JBG et revenir amorce </td></tr><tr><td> Avantage UltraPure PCR dNTP Mix </td><td> Clontech, Mountain View, CA </td><td> 639125 </td><td></td></tr><tr><td> Avantage 2 polymérase </td><td> Clontech, Mountain View, CA </td><td> 639201 639202 ou </td><td> Une bonne relecture, de démarrage à chaud Taq polymérase </td></tr></tbody></table>

a pcr-based genotyping method to distinguish between wild-type and ornamental varieties of imperata cylindrica

Wild-type I. cylindrica (imperata cylindrique) est l&#39;un des dix meilleurs pires plantes envahissantes dans le monde, un impact négatif sur les ressources agricoles et naturelles dans 73 pays différents à travers l&#39;Afrique, en Asie, en Europe, en Nouvelle-Zélande, Océanie et les 1-2 Amériques. Imperata cylindrique se forme rapidement-étalement, stands monodominantes qui déplacent une grande variété d&#39;espèces végétales indigènes et à leur tour menacer les animaux indigènes qui dépendent des espèces de plantes indigènes déplacées pour le fourrage et un abri. Pour ajouter au problème, une variété ornementale [I. cylindrica var. koenigii (Retzius)] est largement commercialisé sous les noms de &#39;Rubra&#39; Imperata cylindrica, Red Baron, et de l&#39;herbe de sang japonais (JBG). Cette variété est présumément stérile et non invasive et est considéré comme une plante ornementale souhaitable pour ses feuilles de couleur rouge. Toutefois, dans des conditions correctes, JBG peut produire des graines viables (Carol Holko, 2009 communication personnelle) et peut revenir à un vert iforme nvasive qui est souvent impossible de distinguer imperata cylindrique comme il faut sur ​​les caractéristiques distinctives de la variété de type sauvage invasive 4 (figure 1). Cela rend l&#39;identification à l&#39;aide la morphologie d&#39;une tâche difficile, même pour les taxonomistes de plantes bien formés. La réversion de la JBG à un phénotype agressif vert n&#39;est pas non plus un phénomène rare. En utilisant des comparaisons de séquences de codage et de régions variables de l&#39;ADN nucléaire et chloroplastique, nous avons confirmé que JBG est revenu à la invasif verte au sein des Etats du Maryland, la Caroline du Sud, et le Missouri. JBG a été vendu et planté dans un état à peu près tous sur le continent américain où il n&#39;ya pas une infestation active imperata cylindrique. La mesure du problème reviendra dans pas bien compris parce que les plantes redevenus sont sans papiers et souvent détruits. L&#39;application de ce protocole fournit une méthode moléculaire pour identifier les JBG revient et peut aider à garder ces variétés de co-produisent unee peut s&#39;hybrider. Imperata cylindrique est une allofécondation obligatoire et, lorsqu&#39;on les croise avec un génotype différent, peut produire dispersées par le vent viable semences qui se propagent sur ​​de grandes distances imperata cylindrique 5-7. JBG a un génotype légèrement différente de celle imperata cylindrique et peut être en mesure de former des hybrides viables avec imperata cylindrique. Pour ajouter au problème, JBG est plus froid et tolérante à l&#39;ombre de imperata cylindrique 8-10, et le flux génétique entre ces deux variétés est susceptible de générer des hybrides qui sont plus agressifs, tolérante à l&#39;ombre, et résistant au froid que de type sauvage imperata cylindrique. Bien que de type sauvage imperata cylindrique infeste actuellement plus de 490 millions d&#39;hectares dans le monde, aux États-Unis du Sud-Est il infeste plus de 500.000 hectares et est capable d&#39;occuper la plupart des Etats-Unis car il se propage rapidement vers le nord en raison de sa niche large et géographique 3,7,11 potentiel. Le potentiel d&#39;un croisement génétique est une grave préoccupation pour le Programme de l&#39;USDA-APHIS fédérale Semaine nocives. Actuellement, l&#39;USDA-APHIS interdit JBG dans les Etats whavant il ya les grands infestations imperata cylindrique (par exemple, en Floride, Alabama, Mississippi). Toutefois, la prévention des deux variétés de la combinaison peut s&#39;avérer plus difficile que imperata cylindrique et JBG élargir leurs distributions. En outre, la répartition de la JBG revenir est actuellement inconnu et sans la capacité d&#39;identifier ces variétés par la morphologie, certains infestations imperata cylindrique peut être le résultat de JBG revient. Malheureusement, les méthodes actuelles d&#39;identification moléculaire s&#39;appuient généralement sur AFLP (amplified fragment length polymorphisms) et séquençage de l&#39;ADN, qui sont tous deux longues et coûteuses. Ici, nous présentons le premier coût-efficace et fiable basée sur la PCR méthode génotypage moléculaire de distinguer avec précision entre les imperata cylindrique et JBG revenir.

Watch this Scientific Journal Video about A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica at JoVE.com

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Nous fournissons un protocole de génotypage coût-efficace et rapide moléculaire qui emploie diverses amorces spécifiques de PCR que les différences cibles séquence d&#39;ADN au sein de la région de l&#39;espaceur chloroplastique trnL-F à la différence entre les variétés de<em&gt; Imperata cylindrica</em&gt; (Imperata cylindrique) qui ne peuvent pas être distingués par la seule morphologie. Ces variétés sont la mauvaise herbe nuisible sur la liste fédérale, imperata cylindrique et étroitement liée, répandue variété ornementale, je<em&gt;. cylindrica</em&gt; Var.<em&gt; Koenigii</em&gt; (Herbe de sang japonais).

Une méthode de génotypage basé sur la PCR pour distinguer les variétés de type sauvage et les plantes ornementales de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

a-pcr-based-genotyping-method-to-distinguish-between-wild-type

Research

JoVE Journal

Biology

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

31.4K vues.  The University of Alabama, Huntsville. Nous fournissons un protocole de génotypage coût-efficace et rapide moléculaire qui emploie diverses amorces spécifiques de PCR que les différences cibles séquence d'ADN au sein de la région de l'espaceur chloroplastique trnL-F à la différence entre les variétés de Imperata cylindrica (Imperata cylindrique) qui ne peuvent pas être distingués par la seule morphologie. Ces variétés sont la mauvaise herbe nuisible sur la liste fédérale...

Vidéo: A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyroséquençage: une méthode simple pour le génotypage précis

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Recherche

Biologie

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Identifier les effets des mutations du gène BRCA1 sur la recombinaison homologue en utilisant les cellules qui expriment endogène de type sauvage du gène BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Essai de superposition des protéines membranaires: Un protocole pour tester l&#39;interaction entre les protéines solubles et insolubles In vitro</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Optimisé basé sur la PCR Détection de Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: un procédé de croissance Arabidopsis Et plants de tomates dans des plaques à 96 puits pour génotypage à haut débit

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

Le génotypage des échantillons de plantes et d&#39;animaux sans purification d&#39;ADN Avant

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

Infinium Assay pour Grands génotypage SNP Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Le poisson zèbre rapide et efficace génotypage utilisant la PCR avec haute résolution Melt analyse

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Analyse qPCRTag - un débit élevé, PCR temps réel pour Sc2.0 Génotypage

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Détermination des acides sialiques dans les échantillons de foie et de lait de type sauvage et<em&gt; CMAH</em&gt; Souris knock-out.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...