Name: a practical and novel method to extract genomic dna from blood collection kits for plasma protein preservation
Uploaded: 2013-05-18T14:00:00
Description: 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

1.. Sample Collection <ol><li> Saml blodprøver fra personer med ordentlig informeret samtykke. For hvert individ, uafgjort 4 til 5 ml blod i en P100 rør (BD Diagnostics, Franklin Lake, NY, USA). Til sammenligningsformål indsamle samtidig blod i et EDTA rør. </li><li> BD P100 rør indeholder 15,8 ml spraytørret K2EDTA og et frysetørret proprietære bredspektret cocktail af proteasehæmmere for at hindre koagulation og stabilisere plasmaproteiner. De indeholder også en mekanisk separator. Efter opsamling af fuldb...

<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>

Mange menneskelige sygdomme har genetiske årsager og udforske det genetiske grundlag for sygdomme hos mennesker kræver en stigende høj kvalitet DNA. Den mest almindelige kilde af DNA, der anvendes i genetiske undersøgelser er antikoaguleret fuldblod. Fordi de fleste kliniske laboratorier udfører biokemiske, serologiske og viral afprøvning som et første skridt i fænotypisk udfald undersøgelse, blodet ofte opsamles i et plasma rør. Efter opsamling af plasmaet, er den komprimerede blod ofte kasseres. Derfor, når...

<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

Laboratorieundersøgelser kan gøres på de cellulære eller flydende dele af blodet. Anvendelsen af ​​forskellige blodopsamlingsrør bestemmer del af blodet, der kan analyseres (fuldblod, plasma eller serum). Laboratorier involveret i at studere den genetiske grundlag af menneskelige lidelser er afhængige af antikoaguleret fuldblod opsamlet i EDTA-holdige Vacutainer som kilde af DNA til genetisk / genomisk analyse. Fordi de fleste kliniske laboratorier udfører biokemiske, serologiske og viral afprøvning som et første skridt i fænotypisk udfald undersøgelse, antikoaguleret blod også opsamlet i heparin-holdige rør (plasma rør). Derfor når DNA og plasma er nødvendige for samtidige og parallelle analyser af både genomiske og proteomiske data, er det almindeligt at samle blod i både EDTA og heparin rør. Hvis blod kan indsamles i et enkelt rør, og tjene som en kilde til både plasma og DNA, ville denne metode blive betragtet som en avancement til eksisterende methods. Brugen af ​​den komprimerede blod efter plasma udvinding repræsenterer en alternativ kilde til genomisk DNA, og dermed minimere mængden af ​​blod behandlede prøver og reducere antallet af nødvendige prøver fra hver patient. Dette ville i sidste ende spare tid og ressourcer. BD P100 blodtapning system til plasmaprotein konservering blev oprettet som en forbedret metode i forhold til tidligere plasma eller serum samling rør 1, for at stabilisere proteinindholdet i blodet, så bedre protein biomarkør opdagelse og proteomics eksperimenter fra humant blod. BD P100 rør indeholder 15,8 ml spraytørret K2EDTA og en frysetørret proprietære bredspektret cocktail af proteaseinhibitorer for at hindre koagulation og stabilisere plasmaproteiner. De omfatter også en mekanisk separator, som giver en fysisk barriere mellem plasma og cellebundfald efter centrifugering. Kun få metoder er blevet udviklet til at udvinde DNA fra størknet blod samples indsamlet i gamle plasma rør 2-4. Udfordringer fra disse metoder blev primært forbundet med den type separator inde i rørene (gel separator) og omfattede vanskeligt inddrive størknet blod, besværet med fragmentering eller dispergering af blodprop, og obstruktion af blodprop ekstraktion ved separation gelen. Vi præsenterer den første metode, som trækker og renser genomisk DNA fra blod trukket i de nye BD P100 rør. Vi sammenligne kvaliteten af ​​DNA-prøven fra P100 rør til at fra EDTA-rør. Vores tilgang er enkel og effektiv. Det indebærer fire store trin som følger: 1) brug af et plasma BD P100 (BD Diagnostics, gnister, MD, USA) rør med mekanisk separator for blodprøvetagning, 2) fjernelse af den mekaniske separator ved hjælp af en kombination af saccharose og en steril papirclips metallisk krog, 3) udskillelse af buffy coat lag indeholdende de hvide celler og 4) isolering af genomisk DNA fraBuffy coat hjælp af en almindelig kommerciel DNA ekstraktion kit eller en lignende standard protokol.

DNA kvalitetsvurdering og koncentrationsmåling Udbytterne af P100_DNAs var betydeligt lavere end EDTA_DNAs (tabel 1 og 2). DNA renheden repræsenteret ved sin 260/280 forholdet værdi er den samme for både P100_DNA og EDTA_DNA prøvesæt (tabel 1 og 2). Kvaliteten af DNA&#39;et er ret ensartet inden for hvert sæt prøve selvom nogle nedbrydning ses i EDTA_DNA prøver, som indikeret af lys smear (figur 2). De...

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

Vi beskriver en ny fremgangsmåde til isolering af genomisk DNA fra fuldblod ved plasma / serologi. Efter plasma kollektion er den komprimerede blodet normalt kasseres. Vores hidtil ukendte fremgangsmåde udgør en betydelig forbedring i forhold til eksisterende metoder og gør DNA og plasma fås fra en enkelt samling, uden at anmode om yderligere blod.

En praktisk og Novel metode til at udvinde Genomisk DNA fra Blood Collection Kits til Plasma Protein 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 ...

Research

JoVE Journal

Biology

Isolation of Genomic DNA from Mouse Tails

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 ...

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 ...

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

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 ...

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 ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Selektiv opsamling af 5-hydroxymethylcytosine fra genomisk 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 ...

A Practical Guide to Phylogenetics for Nonexperts

En praktisk vejledning til fylogeni for Nonexperts

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

Fremgangsmåde til screening og validering af resistente mutationer Against kinaseinhibitorer

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

Gentagen Blodprøvetagning for blodprøver Voksen Zebrafisk

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

Forstærkning, Næste generations sekventering, og Genomisk DNA Kortlægning af retroviral integration steder

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