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。样品采集<ol><li>抽取血液样本，从个人与适当的知情同意。对于每一个人来说，绘制成P100管（BD诊断，富兰克林湖，NY，USA）的4〜5毫升血液。为了便于比较，同时收集血液EDTA管。 </li><li> BD P100管含有15.8毫升的喷雾干燥K2EDTA和冻干专利广谱蛋白酶抑制剂的鸡尾酒，以防止凝血和稳定的血浆蛋白。它们还包含一个机械分离器。采集全血后，保持在室温下放置60分钟至过夜，直到两个管子处理发?...

<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>

许多人类疾病的遗传原因和探索人类疾病的遗传基础，要求越来越高品质的DNA。遗传研究中使用的最常见的来源的DNA抗凝全血。因为大多数临床实验室进行生化，血清学，病毒检测表型结果调查中，作为第一步，血液被收集在一个等离子管。后收集血浆，压实的血液经常被丢弃。因此，当需要的DNA进行基因的研究或测试时，需要一个额外的抽血来自同一病人。丢弃的压实血指DNA的一个潜在来源，?...

<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

实验室测试，可以做到对细胞或血液的流体部分。不同的血液收集管的使用确定的部分可以被分析的血液（全血，血浆或血清）。实验室参与研究人类疾病的遗传基础，依靠抗凝全血收集在含EDTA真空采血管作为源DNA遗传/基因组分析。因为大多数临床实验室进行生化，血清学和病毒的​​检测表型的结果调查作为第一步，也收集在含肝素管（等离子管）抗凝血。因此，当DNA和等离子所需的同时并行分析的基因组和蛋白质组数据，这是习惯EDTA和肝素管中收集血液。如果血液可以在单管中收集作为血浆和脱氧核糖核酸的源，该方法将被认为是提高现有的甲基消耗臭氧层物质。使用压实血提取血浆后表示的另外来源的基因组DNA，从而减少了处理的血液样本量，并减少从每个病人所需的样本的数量。这将最终节省时间和资源。 创建BD P100血液收集系统保存血浆蛋白作为一种改进方法比以前的血浆或血清收集管1，稳定血液中的蛋白质含量，从而更好的蛋白质生物标志物的发现和蛋白质组学实验从人血。 BD P100的管内含有15.8毫升K2EDTA喷雾干燥和冻干的专有广谱蛋白酶抑制剂鸡尾酒，以防止凝血和稳定的血浆蛋白。它们还包括机械分离器，它提供了离心后的血浆和细胞颗粒之间的物理屏障。已经制订了一些方法提取DNA血块SA收集旧的等离子管mples 2-4。这些方法的挑战主要与隔板内部管（凝胶分离器）的类型，包括恢复血液凝块的困难，不便过于分散或分散的血块，血块阻塞提取分离凝胶。 我们提出的第一个方法，在新的BD P100管抽血，基因组DNA的提取和净化。我们的DNA样品的质量进行比较，从P100管从EDTA管。我们的做法是简单而有效的。它包括四个主要步骤如下：1）使用的等离子体BD（BD诊断，火花，马里兰州，美国）P100管机械分离器的采血，2）机械分离器除去使用蔗糖的组合和无菌的曲别针的金属钩，3）的血沉棕黄层的层含有白细胞和4的分离）从基因组DNA的分离白膜使用一个普通的商业DNA提取试剂盒或类似的标准协议。

DNA质量评估和浓度测量的产率P100_DNAs明显低于那些的EDTA_DNAs（ 表1和2）。 260/280的比值所表示的DNA的纯度都P100_DNA EDTA_DNA样品组（ 表1和2）类似。虽然一些退化看到在EDTA_DNA样本，如由光线浸润（ 图2）的各组样本内的DNA的质量是相当均匀的。更多退化的迹象表明EDTA_DNAs也表明DNA浓度降低。 基因分...

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

我们所描述的从全血收集的血浆/血清中分离基因组DNA的新方法。血浆采集后，压实血通常是被丢弃。我们的新方法较现有方法相比，一个显着的改善，使得DNA，不要求额外的血液和血浆可以从一个单一的集合。

一种实用的新的方法来提取基因组DNA保存血浆蛋白从血液采集套件

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

从鼠尾基因组DNA提取

科研

生物学

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

从美国的马蹄蟹，鲎波吕斐摩斯采血车

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

超高分子量基因组DNA的提取土壤和沉积物

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

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

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

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

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

从基因组DNA选择性捕获的5  - 羟甲基

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

实用指南系统发育的非专家

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

对于耐药性突变对激酶抑制剂的筛选和验证方法

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

重复采血车在成年斑马鱼验血

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

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

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