1. Prélèvement <ol><li> Prélever des échantillons de sang de personnes atteintes de consentement éclairé adéquat. Pour chaque individu, tirage 4 à 5 ml de sang dans un tube P100 (BD Diagnostics, le lac Franklin, NY, USA). Aux fins de comparaison, recueillir simultanément sang dans un tube EDTA. </li><li> Le P100 tubes BD contiennent 15,8 ml K2EDTA atomisé et un cocktail à large spectre propriétaire lyophilisée d&#39;inhibiteurs de protéase pour éviter la coagulation et de stabiliser les protéines plasmatiques. Ils contiennent également un séparateur mécanique. Après avoir recueilli le sang total, maintenir les deux tubes à la température ambiante pendant 60 minutes à une nuit jusqu&#39;à ce que la transformation se produit. </li><li> Centrifuger les tubes BD P100 à 2500 g pendant quinze minutes à la température ambiante. Transférer le plasma collecté au-dessus du séparateur mécanique dans des tubes de micro-centrifugation. </li><li> Stocker les tubes P100, contenant le sang compacté sous le séparateur, à -80 ° C jusqu&#39;à ce que vous êtes prêt à commencer l&#39;ADN posteraction. </li></ol> 2.1 Du sang du tube P100 (P100_DNA) <ol><li> Le P100 tubes sont stockés à -80 ° C après extraction du plasma. Dans les 3 à 4 mois, récupérer le tube P100 contenant le sang compacté du congélateur et décongeler à 37 ° C dans un bain-marie pendant 5 min (figure 1A). Retirez tout plasma résiduel qui se trouve au-dessus du séparateur. Ajouter 2 ml de solution de saccharose à 17% (solution de gradient testé dans la maison - données non publiées) dans les tubes P100-dessus du séparateur (figure 1B). Centrifuger les tubes à la température ambiante pendant 20 min à 2500 g, ce procédé pousse le séparateur mécanique à la partie supérieure du tube, et on obtient trois couches d&#39;une solution comprenant la couche leuco-plaquettaire (figure 1C). Extraire manuellement le séparateur à l&#39;aide d&#39;un trombone métallique accroché steriled (figure 1D). </li><li> Après avoir récupéré le séparateur mécanique de chaque tube (figure 1D), enlever et discard de la couche de saccharose haut. Transférer la couche intermédiaire, ce qui représente la couche couche leuco-plaquettaire (BC), dans un tube conique stérile 15 ml. Ajouter tampon phosphate salin (PBS) pour augmenter le volume de la couche leucocytaire à 3 ml. Extraire l&#39;ADN à partir de la couche leuco-plaquettaire en utilisant des réactifs de la trousse de sang Puregene Qiagen selon la recommandation du fabricant, à l&#39;exception d&#39;une vitesse de centrifugation de 2500 g (au lieu de 2.000 g). </li><li> Pour lyse et enlever les globules rouges (RBC), ajouter 9 ml de lyse RBC à 3 ml de couche leuco-plaquettaire. Inversez chaque tube plusieurs fois et incuber à température ambiante pendant 10 min avec inversion occasionnelle pendant l&#39;incubation. Spin chaque mélange vers le bas à 2500 g pendant 5 min et jeter le surnageant. Traiter le culot restant dans chaque tube avec 3 ml d&#39;une solution de lyse des cellules et le vortex pendant 15 s. Traiter le lysat avec 1 ml de solution de précipitation des protéines et vortex pendant 15 sec. </li><li> Centrifugeuse à 2500 g pendant 5 min et transférer le surnageant dans un tube conique de 15 ml contenant fraîche3 ml d&#39;isopropanol à 100%. Inverser les nouveaux tubes 10 fois et centrifuger à 2500 g pendant 3 min. Jeter le surnageant et ajouter 1 ml d&#39;éthanol à 70%. Inverser les tubes coniques à quelques reprises pour déloger et laver le culot d&#39;ADN. Centrifuger à 2500 g pendant 1 min. Laisser le pellet à sécher pendant 3 min (placer le tube à l&#39;envers) à température ambiante, puis suspendre l&#39;ADN avec 250 ml de solution de réhydratation à 37 ° C pendant 10 min et le transfert à un pré-étiquetés 1,7 ml microtubes. Conserver les tubes à -80 ° C jusqu&#39;à utilisation. </li></ol> 2.2 De Sang EDTA Ttube (EDTA_DNA) <ol><li> Le sang total pour les essais d&#39;immuno-routine est recueilli dans des tubes en plastique vaccutainer revêtu par pulvérisation avec 10,8 mg de K 2 EDTA et stockée à -80 ° C. Parce que les tubes ne contiennent pas un séparateur mécanique, l&#39;extraction d&#39;ADN ne comprend pas les étapes impliquant le séparateur mécanique (2.1.1 et 2.1.2). </li><li> Dans les 3 à 4 mois de stockage du sang, le DNA est extrait en utilisant le martin-pêcheur Flex (Thermo Fisher Scientific, Waltham, MA, USA). Il s&#39;agit d&#39;un système d&#39;extraction de l&#39;ADN d&#39;automatisation sur la base de la technologie à poudre magnétique et se compose de lyse / liaison de l&#39;ADN, plusieurs étapes de lavage des cellules et l&#39;élution de l&#39;ADN. </li><li> Le kit utilisé dans l&#39;extraction de l&#39;ADN est le Flex 24 kit de sang de l&#39;ADN Kingfisher (Qiagen, Allemagne). </li></ol> 3. Quantité d&#39;ADN et évaluation de la qualité La quantité, la qualité et l&#39;intégrité de l&#39;ADN génomique ont été évalués par une combinaison de méthode qui inclut PicoGreen, la spectroscopie et l&#39;électrophorèse. <ol><li> La concentration de l&#39;ADN génomique est déterminée par quantification Nanodrop et PicoGreen. </li><li> La pureté est déterminée à partir de la mesure du rapport 260/280 sur le spectrophotomètre Nanodrop. </li><li> L&#39;échantillon (500 ng) est également chargé sur gel d&#39;agarose 1% pour évaluer l&#39;intégrité (dégradation) de l&#39;ADN. </li></ol> 4. Genotyping analyses et contrôle de qualité <ol><li> Cinq échantillons de leurs EDTA_DNA correspondants P100_DNA et sont choisis au hasard pour une analyse plus approfondie en utilisant le Immunochip personnalisé (Immuno analyse ADN BeadChip). Immunochip est une puce de génotypage Infinium contenant 196 524 polymorphismes et est conçu pour les études immunogénétiques 5. </li><li> Pour chaque échantillon, 200 ng d&#39;ADN est amplifié, fragmenté, précipité et remise en suspension dans du tampon d&#39;hybridation appropriée. </li><li> Les échantillons dénaturés sont hybridés sur Immunochips à 48 ° C pendant un minimum de 16 heures. </li><li> Après l&#39;hybridation, les Immunochips sont traitées pour des réactions d&#39;extension d&#39;une seule base et colorées. </li><li> Les immunochips sont ensuite imagées par Illumina HISCAN lecteur d&#39;arrangement de billes et les données d&#39;intensité de talon normalisées sont chargées dans le logiciel Illumina GenomeStudio et convertis en génotypes. Les génotypes sont appelées à l&#39;aide de l&#39;algorithme autocalling de GenomeStudio. Le contrôle de la qualité dansimplique d exclusion des polymorphismes de nucléotide simple (SNP), avec un taux d&#39;appels &lt;95%. </li><li> Le taux d&#39;appel SNP est utilisée comme une mesure de l&#39;efficacité de dosage immunochip. Il est calculé comme le pourcentage du nombre total de dosages sur la puce (196 524 SNP codée sur chaque puce) qui permettent d&#39;atteindre une intensité de signal suffisante et de qualité pour recevoir une assignation de génotype automatisé. On s&#39;attend ADN de haute qualité (260/280 rapport de 1,8 ou plus et en l&#39;absence de dégradation) pour atteindre au moins le même taux d&#39;appel que l&#39;ADN de contrôle HapMap inclus dans l&#39;essai immunochip. Le contrôle de qualité comprend l&#39;exclusion de SNP avec un taux d&#39;appels &lt;95%, dans chaque ensemble de données. </li></ol>

<ol>
	<li>Yi, J., Kim, C., Gelfand, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+intrinsic+proteolytic+activities+moderates+preanalytical+variability+and+instability+of+human+plasma.">Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma.</a> Journal of Proteome Research. 6, 1768-1781 (2007).</li><li>Garg, U. C., Hanson, N. Q., Tsai, M. Y., Eckfeldt, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+method+for+extraction+of+DNA+from+fresh+and+cryopreserved+clotted+human+blood.">Simple and rapid method for extraction of DNA from fresh and cryopreserved clotted human blood.</a> Clinical Chemistry. 42, 647-648 (1996).</li><li>Salazar, L. A., Hirata, M. H., Cavalli, S. A., Machado, M. O., Hirata, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+procedure+for+DNA+isolation+from+fresh+and+cryopreserved+clotted+human+blood+useful+in+clinical+molecular+testing.">Optimized procedure for DNA isolation from fresh and cryopreserved clotted human blood useful in clinical molecular testing.</a> Clinical Chemistry. 44, 1748-1750 (1998).</li><li>Xu, R., Ye, P., Luo, L., Wu, H., Dong, J., Deng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+DNA+purification+from+samples+of+highly+clotted+blood.">A simple and efficient method for DNA purification from samples of highly clotted blood.</a> Molecular Biotechnology. 46, 258-264 (2010).</li><li>Cortes, A., Brown, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promise+and+pitfalls+of+the+Immunochip.">Promise and pitfalls of the Immunochip.</a> Arthritis Research &amp; Therapy. 13, 101 (2011).</li><li>Basuni, A. A., Butterworth, L. A., Cooksley, G., Locarnini, S., Carman, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+extraction+method+from+blood+clots+for+studies+requiring+both+host+and+viral+DNA.">An efficient extraction method from blood clots for studies requiring both host and viral DNA.</a> Journal of Viral Hepatitis. 7, 241-243 (2000).</li><li>Kanai, N., Fujii, T., Saito, K., Tokoyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simple+method+for+preparation+of+genomic+DNA+from+easily+obtainable+clotted+blood.">Rapid and simple method for preparation of genomic DNA from easily obtainable clotted blood.</a> Journal of Clinical Pathology. 47, 1043-1044 (1994).</li><li>Adkins, K. K., Strom, D. A., Jacobson, T. E., Seemann, C. R., O'Brien, D. P., Heath, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+genomic+DNA+purified+from+clotted+blood+samples+for+single+nucleotide+polymorphism+genotyping.">Utilizing genomic DNA purified from clotted blood samples for single nucleotide polymorphism genotyping.</a> Archives of Pathology &amp; Laboratory Medicine. 126, 266-270 (2002).</li><li>Siafakas, N., Burnett, L., Bennetts, B., Proos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonenzymatic+extraction+of+DNA+from+blood+collected+into+serum+separator+tubes.">Nonenzymatic extraction of DNA from blood collected into serum separator tubes.</a> Clinical Chemistry. 41, 1045-1046 (1995).</li><li>Everson, R. B., Mass, M. J., Gallagher, J. E., Musser, C., Dalzell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+cryopreserved+clotted+human+blood.">Extraction of DNA from cryopreserved clotted human blood.</a> BioTechniques. 15, 18-20 (1993).</li><li>Se Fum Wong, S., Kuei, J. J., Prasad, N., Agonafer, E., Mendoza, G. A., Pemberton, T. J., Patel, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+DNA+isolation+from+clotted+blood+extricated+rapidly+from+serum+separator+tubes.">A simple method for DNA isolation from clotted blood extricated rapidly from serum separator tubes.</a> Clin. Chem. 53, 522-524 (2007).</li><li>Garg, U. C., Hanson, N. Q., Tsai, M. Y., Eckfeldt, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+rapid+method+for+extraction+of+DNA+from+fresh+and+cryopreserved+clotted+human+blood.">Simple and rapid method for extraction of DNA from fresh and cryopreserved clotted human blood.</a> Clin. Chem. 42, 647-648 (1996).</li><li>Salazar, L. A., Hirata, M. H., Cavalli, S. A., Machado, M. O., Hirata, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+procedure+for+DNA+isolation+from+fresh+and+cryopreserved+clotted+human+blood+useful+in+clinical+molecular+testing.">Optimized procedure for DNA isolation from fresh and cryopreserved clotted human blood useful in clinical molecular testing.</a> Clin. Chem. 44, 1748-1750 (1998).</li><li>Matheson, L. A., Duong, T. T., Rosenberg, A. M., Yeung, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+sample+collection+and+storage+methods+for+multicenter+immunologic+research+in+children.">Assessment of sample collection and storage methods for multicenter immunologic research in children.</a> Journal of Immunological Methods. 339, 82-89 (2008).</li><li>Xu, R., Ye, P., Luo, L., Wu, H., Dong, J., Deng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+DNA+purification+from+samples+of+highly+clotted+blood.">A simple and efficient method for DNA purification from samples of highly clotted blood.</a> Mol. Biotechnol. 46, 258-264 (2010).</li><li>Polychronakos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+points+in+mapping+autoimmunity.">Fine points in mapping autoimmunity.</a> Nature. 43, 1173-1174 (2011).</li><li>Cooper, J. D., Simmonds, M. J., Walker, N. M., Burren, O., Brand, O. J., Guo, H., Wallace, C., Stevens, H., Coleman, G., Franklyn, J. A., Todd, J. A., Gough, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seven+newly+identified+loci+for+autoimmune+thyroid+disease.">Seven newly identified loci for autoimmune thyroid disease.</a> Human Molecular Genetics. , (2012).</li></ol>

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De nombreuses maladies humaines ont des causes génétiques et explorer la base génétique des maladies humaines exige une augmentation de l&#39;ADN de haute qualité. La source la plus commune de l&#39;ADN utilisée dans les études génétiques anticoagulant du sang total. Parce que la plupart des laboratoires cliniques effectuent des tests biochimiques, sérologiques et virales comme une première étape dans l&#39;enquête de résultat phénotypique, le sang est souvent recueillie dans un tube à plasma. Après le ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>BD P100 tubes</td>
 <td>BD Diagnostics</td>
 <td>366448</td>
 </tr>
 <tr>
 <td>EDTA tubes</td>
 <td>BD Diagnostics</td>
 <td>367863</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378</td>
 </tr>
 <tr>
 <td>Paper clip</td>
 <td>Office product</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Corning</td>
 <td>430052</td>
 </tr>
 <tr>
 <td>Red blood cells (RBC)</td>
 <td>Qiagen</td>
 <td>158389 (kit)</td>
 </tr>
 <tr>
 <td>Phosphate Buffer Saline (PBS)</td>
 <td>Thermo Scientific</td>
 <td>SH30256.01</td>
 </tr>
 <tr>
 <td>BioSprint 96 DNA Blood Kit (48) </td>
 <td>Qiagen</td>
 <td>940054</td>
 </tr>
 <tr>
 <td>1.7 ml microtubes</td>
 <td>Axygen</td>
 <td>MCT-175-C</td>
 </tr>
 <tr>
 <td>Human Immuno DNA Analysis BeadChip Kit</td>
 <td>Illumina</td>
 <td>WG-352-1001</td>
 </tr>
 <tr>
 <td>Bead array reader</td>
 <td>Illumina</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>GenomeStudio Software</td>
 <td>Illumina</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

a practical and novel method to extract genomic dna from blood collection kits for plasma protein preservation

Les tests de laboratoire peuvent être effectués sur les parties cellulaires ou de liquide du sang. L&#39;utilisation de différents tubes de prélèvement de sang détermine la portion du sang pouvant être analysé (sang total, le plasma ou le sérum). Les laboratoires impliqués dans l&#39;étude de la base génétique des maladies humaines s&#39;appuient sur sang total anticoagulant recueillies dans Vacutainer contenant de l&#39;EDTA comme source d&#39;ADN pour analyse génétique / génomique. Parce que la plupart des laboratoires cliniques effectuent des tests biochimiques, sérologiques et virales comme une première étape dans l&#39;enquête de résultat phénotypique, le sang coagulé sont aussi recueillis dans un tube contenant de l&#39;héparine (tube à plasma). Par conséquent, lorsque l&#39;ADN et de plasma sont nécessaires pour des analyses simultanées et parallèles de données à la fois génomiques et protéomiques, il est de coutume pour recueillir le sang dans les deux tubes EDTA et l&#39;héparine. Si le sang peut être recueilli dans un seul tube et servir de source pour le plasma et l&#39;ADN, cette méthode serait considérée comme un avancement à l&#39;existant methSAO. L&#39;utilisation du sang compactée après l&#39;extraction du plasma représente une autre source d&#39;ADN génomique, ce qui réduit la quantité d&#39;échantillons sanguins traitées et de réduire le nombre d&#39;échantillons requis pour chaque patient. Ce serait finalement gagner du temps et des ressources. Le système de collecte de sang P100 BD pour la préservation de protéines plasmatiques a été créé comme un procédé amélioré au plasma précédente ou la collecte du sérum tubes 1, pour stabiliser la teneur en protéines du sang, ce qui permet une meilleure découverte des biomarqueurs protéiniques et protéomique expérimentation à partir de sang humain. Le P100 tubes BD contiennent 15,8 ml de K2EDTA atomisé et un cocktail à large spectre propriétaire lyophilisée d&#39;inhibiteurs de protéase pour éviter la coagulation et de stabiliser les protéines plasmatiques. Elles comprennent également un séparateur mécanique, ce qui constitue une barrière physique entre le plasma et les culots cellulaires après centrifugation. Peu de méthodes ont été mises au point pour extraire l&#39;ADN à partir de sang coagulé SAmples recueillis dans des tubes de plasma vieux 2-4. Défis de ces méthodes ont été principalement associés avec le type de séparateur à l&#39;intérieur des tubes (séparateur gel) et comprenaient des difficultés à récupérer le sang coagulé, les inconvénients de la fragmentation ou la dispersion du caillot et l&#39;obstruction de l&#39;extraction caillot par le gel de séparation. Nous présentons la première méthode qui extrait et purifie l&#39;ADN génomique à partir de sang prélevé dans les nouveaux tubes P100 BD. Nous comparons la qualité de l&#39;échantillon d&#39;ADN de P100 tubes à celle des tubes EDTA. Notre approche est simple et efficace. Elle comporte quatre grandes étapes, comme suit: 1) l&#39;utilisation d&#39;un plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tubes avec séparateur mécanique pour la collecte de sang, 2) la suppression du séparateur mécanique à l&#39;aide d&#39;une combinaison de saccharose et un stérile trombone métallique crochet, 3) la séparation de la couche leuco-plaquettaire contenant les globules blancs et 4) l&#39;isolement de l&#39;ADN génomique à partir de l&#39;couche leuco-plaquettaire en utilisant un kit d&#39;extraction d&#39;ADN commercial régulier ou d&#39;un protocole standard similaire.

évaluation de la qualité de l&#39;ADN et la mesure de la concentration Les rendements des P100_DNAs étaient significativement inférieurs à ceux des EDTA_DNAs (tableaux 1 et 2). La pureté d&#39;ADN représentée par sa valeur du rapport 260/280 est similaire pour les deux P100_DNA et ensemble d&#39;échantillons EDTA_DNA (tableaux 1 et 2). La qualité de l&#39;ADN est assez uniforme dans chaque ensemble de l&#39;échantill...

Watch this Scientific Journal Video about A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation at JoVE.com

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Nous décrivons une nouvelle méthode d&#39;isolement de l&#39;ADN génomique à partir de sang total prélevé pour plasma / sérologie. Après la collecte de plasma, le sang compacté est habituellement jeté. Notre nouvelle méthode représente une amélioration significative par rapport aux méthodes existantes et rend l&#39;ADN et plasma disponibles à partir d&#39;une collection unique, sans demander de sang supplémentaire.

Une méthode pratique et roman d&#39;extraire l&#39;ADN génomique à partir de kits de prélèvement sanguin pour la préservation Plasma Protein

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

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41.9K vues.  Emory University School of Medicine and Children's Health Care of Atlanta. Nous décrivons une nouvelle méthode d'isolement de l'ADN génomique à partir de sang total prélevé pour plasma / sérologie. Après la collecte de plasma, le sang compacté est habituellement jeté. Notre nouvelle méthode représente une amélioration significative par rapport aux méthodes existantes et rend l'ADN et plasma disponibles à partir d'une collection unique, sans demander de sang supplémentaire.

Vidéo: A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Isolation of Genomic DNA from Mouse Tails

Isolement de l&#39;ADN génomique de souris Tails

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Biologie

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Collecte de sang du crabe américaine Horseshoe, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extraction d&#39;ADN de haut poids moléculaire génomique par les sols et sédiments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Chromatographie d&#39;affinité lipidique des vésicules médiée utilisant magnétique tri cellulaire activé (LIMACS): une nouvelle méthode pour analyser les interactions protéines-lipides

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Genomic IRM - une ressource publique pour l&#39;étude des schémas de séquence dans l&#39;ADN génomique

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Capture d&#39;sélectif de 5-hydroxyméthylcytosine partir de l&#39;ADN génomique

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Un guide pratique pour Phylogenetics pour non-experts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Un procédé de criblage et de validation des mutations de résistance aux inhibiteurs de kinase

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Collecte de sang répétées pour des tests sanguins dans le poisson zèbre adulte

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplification, Séquençage de nouvelle génération, et l&#39;ADN génomique Cartographie des rétrovirales Sites d&#39;intégration

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...