1. Raccolta dei campioni <ol><li> Raccogliere campioni di sangue di soggetti con adeguata consenso informato. Per ogni individuo, trarre 4-5 ml di sangue in una provetta P100 (BD Diagnostics, Franklin Lake, NY, USA). A scopo di confronto, raccogliere contemporaneamente il sangue in una provetta con EDTA. </li><li> I tubi P100 BD contengono 15,8 ml K2EDTA essiccato a spruzzo e un liofilizzato proprietaria ampio spettro cocktail di inibitori di proteasi per prevenire la coagulazione e stabilizzare le proteine ​​plasmatiche. Essi contengono anche un separatore meccanico. Dopo aver raccolto il sangue intero, mantenere entrambi i tubi a temperatura ambiente per 60 min a tutta la notte fino al verificarsi di elaborazione. </li><li> Centrifugare le provette BD P100 a 2.500 g per quindici minuti a temperatura ambiente. Trasferire il plasma raccolto sopra il separatore meccanico in provette da centrifuga micro. </li><li> P100 Conservare le provette, contenente il sangue compattato sotto il separatore, a -80 ° C fino a quando si è pronti per iniziare il DNA extraction. </li></ol> 2.1 Dal sangue di P100 tubo (P100_DNA) <ol><li> Le P100 tubi sono conservati a -80 ° C dopo l&#39;estrazione del plasma. Entro 3 a 4 mesi, recuperare la provetta contenente il sangue P100 compattato dal congelatore e scongelare a 37 ° in un bagno d&#39;acqua per 5 minuti (Figura 1A). Rimuovere eventuali residui di plasma che si trova sopra il separatore. Aggiungere 2 ml di soluzione di saccarosio al 17% (soluzione gradiente testato in casa - dati non pubblicati) nelle P100 tubi sopra il separatore (Figura 1B). Centrifugare le provette a temperatura ambiente per 20 min a 2500 g; questo processo spinge il separatore meccanico per la parte superiore del tubo e produce tre strati di soluzione compreso il buffy coat (Figura 1C). Estrarre manualmente il separatore utilizzando una graffetta metallica uncinata steriled (Figura 1D). </li><li> Dopo aver recuperato il separatore meccanico da ogni tubo (Figura 1D), rimuovere e discard dello strato di saccarosio superiore. Trasferire lo strato centrale, che rappresenta il livello di buffy coat (BC), in una sterile 15 ml tubo conico. Aggiungere Tampone fosfato salino (PBS) per aumentare il volume buffy coat a 3 ml. Estrarre il DNA dal buffy coat utilizzando reagenti dalla Qiagen Puregene Kit Sangue come da indicazioni del produttore, eccetto una velocità di centrifugazione di 2500 g (invece di 2.000 g). </li><li> Per lisare e rimuovere i globuli rossi (RBC), aggiungere 9 ml di RBC lisi a 3 ml di buffy coat. Capovolgere ogni provetta diverse volte e incubare a temperatura ambiente per 10 min, con occasionale invertente durante l&#39;incubazione. Spin ogni miscela fino a 2.500 g per 5 minuti e scartare il surnatante. Trattare il restante pellet in ogni provetta con 3 ml di soluzione di lisi cellulare e vortex per 15 sec. Trattare il lisato con 1 ml di soluzione di precipitazione proteica e vortex per 15 sec. </li><li> Centrifugare a 2500 g per 5 minuti e trasferire il surnatante in una nuova provetta conica da 15 ml contenente3 ml di isopropanolo al 100%. Invertire i nuovi tubi di 10 volte e centrifugare a 2500 g per 3 min. Eliminare il supernatante e aggiungere 1 ml di etanolo al 70%. Invertire i tubi conici un paio di volte per rimuovere e lavare il pellet di DNA. Centrifugare a 2500 g per 1 min. Lasciar seccare il precipitato per 3 min (posizionare il tubo capovolto) a temperatura ambiente e quindi sospendere il DNA con 250 ml di soluzione di reidratazione a 37 ° C per 10 min e il trasferimento ad un pre-etichettato 1,7 ml provetta. Conservare le provette a -80 ° C fino all&#39;utilizzo. </li></ol> 2.2 da sangue di EDTA Ttube (EDTA_DNA) <ol><li> Il sangue intero per i test di routine immunoematologia viene raccolto in provette di plastica vaccutainer spruzzo-rivestito con 10,8 mg di K 2 EDTA e conservati a -80 ° C. Poiché i tubi non contengono un separatore meccanico, l&#39;estrazione del DNA non include le fasi che coinvolgono il separatore meccanico (2.1.1 e 2.1.2). </li><li> Entro 3-4 mesi di conservazione del sangue, il DNA viene estratto utilizzando il martin pescatore Flex (Thermo Fisher Scientific, Waltham, MA, USA). Si tratta di un sistema di estrazione del DNA di automazione basati su tecnologia di particelle magnetiche ed è costituito da cellule di lisi / DNA vincolanti, diverse fasi di lavaggio e di eluizione del DNA. </li><li> Il kit utilizzato per l&#39;estrazione del DNA è il martin pescatore Flex 24 DNA del sangue kit (Qiagen, Germania). </li></ol> 3. DNA Quantità e valutazione della qualità La quantità, la qualità e l&#39;integrità del DNA genomico sono stati valutati da una combinazione di metodo che comprende PicoGreen, spettroscopia e elettroforesi. <ol><li> La concentrazione del DNA genomico è determinata dalla quantificazione Nanodrop e PicoGreen. </li><li> La purezza è determinata dalla misurazione 260/280 rapporto sullo spettrofotometro Nanodrop. </li><li> Il campione (500 ng) è anche caricato su gel di agarosio 1% per valutare l&#39;integrità (degradazione) del DNA. </li></ol> 4. Genotyping saggi e controllo di qualità <ol><li> Cinque P100_DNA e loro corrispondenti campioni EDTA_DNA sono scelti a caso per ulteriori analisi utilizzando la Immunochip personalizzato (Immuno DNA Analysis BeadChip). Immunochip è un chip di genotipizzazione Infinium contenente 196.524 polimorfismi ed è progettato per gli studi immunogenetici 5. </li><li> Per ciascun campione, 200 ng di DNA è amplificato, frammentata, precipitato e risospeso in tampone di ibridazione appropriate. </li><li> I campioni denaturati sono ibridate su Immunochips a 48 ° C per un minimo di 16 ore. </li><li> Dopo l&#39;ibridazione, i Immunochips sono trattati per le reazioni di estensione singolo-base e macchiati. </li><li> Le immunochips vengono poi ripreso con lettore di serie Illumina HISCAN Bead ei dati di intensità di perline normalizzati vengono caricati nel software Illumina GenomeStudio e convertiti in genotipi. I genotipi sono chiamate utilizzando l&#39;algoritmo autocalling di GenomeStudio. Il controllo di qualità individeranno in esclusione di polimorfismi a singolo nucleotide (SNPs), con un tasso di chiamata &lt;95%. </li><li> Il tasso di chiamata SNP è utilizzato come misura dell&#39;efficienza dosaggio immunochip. Esso è calcolato come percentuale del numero totale di saggi sul chip (196.524 SNP codificati su ogni chip) che realizzano sufficiente intensità e qualità del segnale per ricevere un&#39;assegnazione genotipo automatizzato. DNA di alta qualità (260/280 rapporto di 1,8 o superiore e assenza di degradazione) è previsto per conseguire almeno lo stesso tasso di chiamata come il DNA di controllo HapMap inclusa nel saggio immunochip. Il controllo di qualità comporta l&#39;esclusione di SNPs con un tasso di chiamata &lt;95% in ogni set di dati. </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>

Nessun conflitto di interessi dichiarati.

Molte malattie umane hanno cause genetiche ed esplorare la base genetica delle malattie umane richiede una crescente DNA di alta qualità. La fonte più comune di DNA utilizzato in studi genetici è coagulato sangue intero. Poiché laboratori più eseguire test biochimici clinici, sierologici, e virali come primo passo in esito indagine fenotipica, il sangue viene spesso raccolta in un tubo plasma. Dopo la raccolta del plasma, il sangue compattato viene spesso scartato. Pertanto, quando il DNA è necessario per condurre...

<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

Test di laboratorio possono essere praticati sulle porzioni cellulari o fluido del sangue. L&#39;uso di diverse provette determina la porzione del sangue che può essere analizzato (sangue intero, plasma o siero). Laboratori coinvolti nello studio delle basi genetiche delle malattie umane si basano su anticoagulante del sangue intero prelevato in EDTA contenente vacutainer come fonte di DNA per l&#39;analisi genetica / genomica. Perché laboratori clinici di eseguire più test biochimici, sierologici e virali come un primo passo in un&#39;indagine risultato fenotipico, il sangue coagulato è anche raccolto in provetta contenente eparina (tubo plasma). Pertanto quando sono necessarie DNA e plasma per analisi simultanea e parallela di dati sia genomici e proteomici, è consuetudine per raccogliere il sangue in EDTA e sia con eparina. Se sangue potrebbe essere raccolto in un unico tubo e servire come fonte sia per plasma e DNA, tale metodo sarebbe considerato un avanzamento a met esistentiods. L&#39;utilizzo del sangue compattato dopo estrazione plasma rappresenta una fonte alternativa per il DNA genomico, minimizzando così la quantità di campioni di sangue trasformati e riducendo il numero di campioni richiesti da ogni paziente. Ciò finirebbe per risparmiare tempo e risorse. Il P100 sistema di raccolta del sangue BD per la conservazione delle proteine ​​del plasma sono stati creati come un metodo migliorato nel corso del plasma precedente o raccolta siero tubi 1, per stabilizzare il contenuto proteico del sangue, consentendo una migliore proteina scoperta di biomarcatori e proteomica sperimentazione dal sangue umano. I tubi P100 BD contengono 15,8 ml di K2EDTA essiccato a spruzzo e un liofilizzato proprietaria ampio spettro cocktail di inibitori di proteasi per prevenire la coagulazione e stabilizzare le proteine ​​plasmatiche. Essi includono anche un separatore meccanico, che fornisce una barriera fisica tra plasma e pellet cellulari dopo centrifugazione. Pochi metodi sono stati studiati per estrarre il DNA da sangue coagulato SAmples raccolti nei vecchi tubi di plasma 2-4. Sfide da questi metodi sono stati principalmente associati con il tipo di separatore all&#39;interno dei tubi (separatore di gel) ed inclusi difficoltà di recupero del sangue coagulato, l&#39;inconveniente di frammentare o disperdendo il coagulo e ostruzione del coagulo estrazione dal gel di separazione. Vi presentiamo il primo metodo che estrae e purifica il DNA genomico da sangue prelevato nei nuovi P100 tubi BD. Si confronta la qualità del campione di DNA da P100 tubi, a quella in provette EDTA. Il nostro approccio è semplice ed efficiente. Si tratta di quattro fasi principali, come segue: 1) l&#39;uso di un BD P100 plasma (BD Diagnostics, Sparks, MD, USA) tubo con separatore meccanico per la raccolta del sangue, 2) la rimozione del separatore meccanico utilizzando una combinazione di saccarosio e di un sterile graffetta gancio metallico, 3) la separazione dello strato di buffy coat contenente le cellule bianche e 4) l&#39;isolamento del DNA genomico dalbuffy coat utilizzando un kit di estrazione del DNA normale commerciale o un protocollo standard simile.

Valutazione della qualità del DNA e la misura della concentrazione Le rese di P100_DNAs erano significativamente inferiori a quelli di EDTA_DNAs (Tabelle 1 e 2). La purezza del DNA rappresentata dal valore 260/280 rapporto è simile per entrambi P100_DNA e set di esempio EDTA_DNA (Tabelle 1 e 2). La qualità del DNA è abbastanza uniforme all&#39;interno di ogni serie di campioni sebbene alcuni degradazione è visto nei campio...

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

Stiamo descrivendo un nuovo procedimento per isolare DNA genomico da sangue intero prelevato per plasma / sierologia. Dopo la raccolta del plasma, il sangue compattato viene solitamente scartata. Il nostro nuovo metodo rappresenta un miglioramento significativo rispetto ai metodi esistenti e rende il DNA e plasma disponibili da una singola collezione, senza richiedere ulteriore sangue.

Un metodo pratico e innovativo per estrarre il DNA genomico da kit di raccolta del sangue per Plasma Proteina Preservation

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 ...

a-practical-novel-method-to-extract-genomic-dna-from-blood-collection

Research

JoVE Journal

Biology

41.9K Views.  Emory University School of Medicine and Children's Health Care of Atlanta. Stiamo descrivendo un nuovo procedimento per isolare DNA genomico da sangue intero prelevato per plasma / sierologia. Dopo la raccolta del plasma, il sangue compattato viene solitamente scartata. Il nostro nuovo metodo rappresenta un miglioramento significativo rispetto ai metodi esistenti e rende il DNA e plasma disponibili da una singola collezione, senza richiedere ulteriore sangue.

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

Isolation of Genomic DNA from Mouse Tails

Isolamento del DNA genomico da Tails mouse

Ricerca

Biologia

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.

Raccolta di sangue dal Granchio Ferro di Cavallo Americano, Limulus Polifemo

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.

Estrazione di DNA ad alta peso molecolare genomico da terreni e sedimenti

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.

Vescicole lipidiche-mediata Cromatografia di affinità mediante Magnetic separazione delle cellule attivate (LIMACS): un nuovo metodo per l&#39;analisi delle interazioni proteina-lipide

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.

Genomica risonanza magnetica - una risorsa pubblica per lo Studio Patterns sequenza all&#39;interno del DNA genomico

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.

Cattura selettiva di 5 hydroxymethylcytosine dal DNA genomico

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;

Una guida pratica per Phylogenetics per i non esperti

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 metodo per lo screening e validazione di mutazioni resistenti contro 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 ...

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.

Ripetuto il prelievo di sangue per esami del sangue in Zebrafish adulti

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.

Amplificazione, sequenziamento di prossima generazione, e DNA genomico mappatura dei siti di integrazione retrovirali

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