我们要感谢为加拿大创新，基因组不列颠哥伦比亚省，不列颠哥伦比亚省知识发展基金和不列颠哥伦比亚省森林部和范围基金会，用于支持正在进行的研究森林的土壤生产力。 SL是由微生物多样性和进化图拉基金会资助中心的奖学金支持。

第一部分：DNA的提取 程序 1。 8小时是必需的，用于处理6个样品，不包括DNA定量。 在您开始之前提取，你将需要预先寒意砂浆，杵和抹刀在-20 ° C冰箱或使用液态氮。此外，前寒意50毫升20毫升氯仿异戊醇（24:1）包含在冰上管。设置杂交炉至65 ° C预热。检查CTAB溶液，如果它是结晶温暖至60 ° C至融化的晶体。完成加入，最后的成分，只是在开始之前提取（见下文配方）的变性液和提取液。在通风橱中进行所有提取程序。 2。准备变性缓冲液和提取液。 注：为了最大限度的DNA产量，关键是精确地按照指示的缓冲区。 3。将在预冷迫击炮2克的土壤。 注意：应在现场收集的土壤干冰冷冻运输和储存在-80 ° C解冻和使用DNA提取前2毫米筛筛土。您可以增加土壤的起点金额高达10克 4。添加1毫升变性液对土壤的。 5。冻结和研磨液ñ的土壤 2使用杵，直到土壤颗粒看上去粉状和同质。重复冻结和磨两次。 注：冻结期间保持土壤样品的研磨，用足够的液氮，全面覆盖的土壤样品。在研磨过程中的轻微融化是可以接受的。 6。泥土转移至50 ml锥形管，使用前冷冻锅铲。 注：可以储存在-80 ° C在这一点上，如有必要，地面土壤样品。 7。加入9毫升提取液。要混合的解决方案和土壤，在低速旋涡简要。 注：为了确保缓冲区是均匀的，温暖的，在60 °为10-15分钟，使用前彗星。整个提取其余的缓冲区保持在60 ° C。旋涡速度8（其中10是最大规模）。 8。孵育40分钟，在65℃杂交炉。在孵化过程中，不断旋转管的最低速度，或轻轻反转管每10分钟。 注：水平放置在杂交炉管，让缓冲区接触比表面积大的土壤。该解决方案孵化过程中可能看起来有点粘稠。 9。离心1800 × 克 ，10分钟在10 - 14 ° C上清转移到预冷含20毫升氯仿异戊醇（24:1）50 ml锥形管，置于冰上。 注意：当你转移上清，要小心，不要扰乱有机层（底层）。如果它是必要的，以便只收集干净上清，留下1-2毫升上清。你经常可以看到一个米色上清顶部层（约1-3毫米的厚度）。收集上清液时，请勿打扰这一层。尝试幻灯片沿管壁的提示，以防止本米色层破损。如果上清太浑浊，重复的氯仿 - 异戊醇提取上清液一次。不要使用一次性塑料吸液管转移氯仿，氯仿将熔化的塑料。使用的玻璃吸管，或玻璃量筒，或直接倒入50 ml锥形管氯仿。 10。土壤颗粒提取2次以上; 10.1。加入5 ml提取缓冲液中的土壤颗粒，轻轻地混合使用1毫升的小费在低速短暂的震荡。 注意：摇晃和混合使用前的缓冲区。 10.2。孵育在65℃杂交炉中10分钟。离心1800 × 克，10分钟在10 - 14 ° C 转移上清管在第9步。保持管冰在提取过程中收集上清和氯仿 - 异戊醇。 注意：请确保您添加混合样本之间无交叉上清同一管。 10.3％。重复9.1和9.2有更多的时间。 11。使用一个旋转盘，很轻轻摇动试管，其中包含所收集的上清（14-19毫升）和氯仿 - 异戊醇1.25 10分钟的转速。 注：盖上盖子，非常严密，防止泄漏。奠定了纸巾上的摇板，然后放置在管，所以可以很容易地检测到任何泄漏。 12。离心机在1800 × G，在10分钟20 - 14 ° C。 13。水相转移到一个新的管。 注：请勿打扰之间的水相（顶层）和有机层（底部相间层），当您将上清转移。离开约1.5 - 2毫升上清，以确保只有清洁上清的收集。 14。恢复使用异丙醇（步骤15.a - 21.a）的DNA。另外，使用Amicon ®超15离心过滤装置（步骤15.b - 21.b）如果你希望生物质在你的样品量非常低，这将产生异丙醇沉淀，难以收回几乎没有可见的颗粒。 15.a.将上清液到一个新的管超速离心机。 16.a.添加异丙醇在体积比为0.6:1的收集上清。反转管轻轻几次混合。 17.a在室温下30分钟的孵育。 18.a.离心16000 X克，20分钟20 - 25 ° C。确保所有管的重量是平衡的离心步骤之前。 注：马克管离心力的方向，所以你可以检测出DNA沉淀时更容易DNA产量低。 19.a.取出调迁或真空抽吸异丙醇。小心不要打扰DNA沉淀。 注：尽快删除异丙醇，离心完成。要非常小心，不要失去DNA沉淀。 DNA沉淀的大小可以有很大的不同取决于土壤类型，从清晰可见（〜9毫米直径），难以看清。这是建议由吸痰沿管壁小心，以免吸出的DNA沉淀，去除残留的异丙醇。 20.a.在室温下干燥DNA沉淀5 - 20分钟。 注意：不要超过干燥DNA沉淀，否则将难以溶解的DNA。 21.a.在200μLTE重悬DNA沉淀。温柔攻混合。 DNA溶液转移到1.5 ml管。管保持在4 ° C过夜，以充分溶解DNA。 注：根据颗粒大小，可以调整的TE量添加。一般200 - 400μLTE的效果很好。 *或者按照下列步骤15.b - 21.b. 15.b.预洗Amicon ®超15离心过滤装置，设备和离心机3500 X克中添加15毫升TE为10分钟。丢弃的流量通过。 16.b.将DNA溶液从第13步，预洗Amicon过滤。离心机在3500 X克，直到DNA的体积缩小至200 - 500μL 。丢弃的流量通过。 17.b.两次DNA用TE洗净;添加10毫升TE Amicon过滤装置。在3500 X克离心机，直到DNA量减少约200μL。丢弃的流量通过。重复一次。 18.b. DNA溶液过滤器传输到一个新的1.5 ml管。 19.b.添加40μLTE Amicon过滤。移液器向上和向下，为了恢复在过滤器上的任何DNA残留清洗滤膜两侧。这种洗涤液管一步18.b. 20.b.如果有必要，DNA可进一步由单片机® Ultracel YM - 30离心式过滤器单元的集中;预洗7分钟10000 ×克加入200μLTE和离心过滤器。将DNA溶液，过滤器，并在5000 X克离心1分钟。直到DNA溶液的过滤器上的金额减少约50μL，重复离心。 注：单片机过滤器的最大初始样本量为500μL。不要离心速度超过14000小工直到完全膜干燥不超过离心机。确保一些液体仍然涵盖的过滤器，离心后。如果在离心膜干燥，然后加入15μLTE，搅拌30秒轻轻，并继续加强21.b. 21.b.将过滤装置，倒挂在一个新的单片机管，并在1000 3分钟恢复的DNA XG离心机。 22。量化TAE凝胶DNA（0.5xTAE，0.8％，为4小时50 V），并通过Nanodrop分光光度计或碧绿色的检测和酶标仪。 注：估计DNA的量可以通过比较与已知浓度对凝胶（高分子DNA标记或λHindIII）的样品带，标记带。 23。检查的超高分子量1％，1xTAE凝胶使用脉冲场凝胶电泳（PFGE）（更多细节见步骤二，在第2部分DNA的<a href="https://www.jove.com/index/details.stp?id=1387">完整性</a> https://www.jove.com/index/details.stp?id <a href="https://www.jove.com/index/details.stp?id=1387">= 1387</a> ，DOI：10.3791/1387 2009年Taupp等） 。 注意：您可以使用这代替的低熔点琼脂糖QC目的普通琼脂糖凝胶。总之，电泳条件如下：2-250 KB，6伏初始开关时间：5秒，最后的开关时间：15秒，12小时运行在14 ° C。 SYBR黄​​金染色一小时，可视化的DNA凝胶。 24。如果提取的DNA分子量是令人满意的，然后进行以下CsCl梯度离心步骤。另外，DNA可以储存在-20 ° C或-80 ° C之前，氯化铯离心。 的DNA中位数的大小应等于或超过36 KB，如果你想建造一个大型的插入宏基因组DNA文库。 第二部分。 DNA纯化用氯化铯梯度离心离心制备：1.5 - 2小时 离心步骤：18小时 卸下溴化乙锭（ETBR）和集中DNA离心后：3小时 程序 有关详细信息，请参阅 Wright等。 2009年<a href="https://www.jove.com/index/details.stp?id=1352">https://www.jove.com/index/details.stp?id=1352</a> ，DOI：10.3791/1352 代表性的成果/成果从2克（海外地平线）林土壤有机和矿物的视野（AE，AB和转Bt基因视野）（从长期土壤生产力（LTSP）位于加拿大不列颠哥伦比亚省Skulow湖，现场采样分离出基因组DNA的总量由卑诗省森林部和范围管理）从10毫克至180毫克不等。孤立的DNA中位数的大小通常在40 - 60 KB（图1）之间。在260 nm/280 nm的吸光度比例1.3-1.6之间，峰值在230 nm有一个，这可能表明腐殖酸污染往往观察到的土壤样品。 CsCl梯度离心大大减少这种污染，也增加了260/280的比例为1.8 - 1.9图2A和2B所示。 <img src="files/ftp_upload/1569/1569fig1.jpg" alt="图1" /> 图1。PFGE图片前铯超速离心法提取基因组DNA。 lane1：1 KB标志100纳克，泳道2：λHind三标记100纳克，泳道3：λHind三标记250纳克，泳道4：λHindIII标志500毫微克，lane5：Fosmid 36 KB标记100吴巷6和7：基因组从矿物土层AE巷8和9中分离DNA：从矿物土层的AB孤立的基因组DNA在位于公元前Skulow湖，LTSP网站<img src="/files/ftp_upload/1569/1569fig2tmb.jpg" alt="图2" border="0" /> 图2。Chromograms基因组DNA的检测分光光度计前（A）和氯化铯超速离心（二）后。请注意在铯超速离心法，这表明在基因组DNA质量的改善后在230 nm波长的吸光度值减少。请<a href="http://jove.com/files/ftp_upload/1569/1569fig2.jpg">点击这里</a>看大图图2。

<ol>
	<li>Hurt, R. A., Qiu, X., Wu, L., Roh, Y., Palumbo, A. V., Tiedje, J. M., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recovery+of+RNA+and+DNA+from+soils+and+sediments.">Simultaneous recovery of RNA and DNA from soils and sediments.</a> AEM. 67 (10), 4495-4503 (2001).</li><li>Wright, J. J., Lee, S., Zaikova, E., Walsh, D. A., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+extraction+from+0.22+&#956;M+Sterivex+filters+and+cesium+chloride+density+gradient+centrifugation.">DNA extraction from 0.22 &#956;M Sterivex filters and cesium chloride density gradient centrifugation.</a> JoVE. , (2009).</li><li>Taupp, M., Lee, S., Hawley, A., Yang, J., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+insert+environmental+genomic+library+production.">Large insert environmental genomic library production.</a> J Vis Exp. , (2009).</li></ol>

配方 *变性液 4个M异硫氰酸胍 10毫米的Tris - HCl（pH值7.0） 1毫米EDTA 0.5％2 - 巯基乙醇<table border="0" style="font-size:9pt; width:400px;"><tr><td colspan="2"> 变性的解决方案：共计10毫升 </td></tr><tr><td>异硫氰酸胍（兆瓦118.16） </td><td> 4.73摹</td></tr><tr><td> 1M的Tris - HCl（pH值7.0） </td><td> 100μL </td></tr><tr><td> 0.5M EDTA </td><td> 20μL </td></tr><tr><td colspan="2">加水共9.95毫升。 </td></tr><tr><td colspan="2">高压灭菌。 </td></tr><tr><td colspan="2">使用前加入50μL的2 -巯基乙醇。 （5μL2 - 巯基乙醇，每1毫升变性液） 注：变性液保持在4 ° C。不要使用超过一个星期的缓冲。如果可能的话，请使用新鲜的缓冲。 </td></tr></table> **提取缓冲液 100毫米磷酸钠缓冲液pH值7.0] 100毫米的Tris - HCl [pH值7.0] 100毫米EDTA [pH值8.0] 1.5 M氯化钠 1％十六烷基铵（CTAB） 2％SDS <table border="0" style="font-size:9pt; width:400px;"><tr><td colspan="2"> 1 L总的提取缓冲液</td></tr><tr><td> 1M磷酸钠缓冲液pH值7.0] * </td><td> 100毫升</td></tr><tr><td> 1M的Tris - HCl [pH值7.0] </td><td> 100毫升</td></tr><tr><td> 0.5M EDTA [pH值8.0] </td><td> 200毫升</td></tr><tr><td> 5 M氯化钠</td><td> 300毫升</td></tr><tr><td colspan="2">高压灭菌，并保持在室温。 </td></tr><tr><td colspan="2">添加200毫升，5％十六烷基三甲基溴化铵（CTAB法，高压灭菌）和100毫升，20％SDS（蒸压）之前使用。如果CTAB的结晶，融于60 ° C。 * 1M钠磷酸盐缓冲液：57.7毫升，1M钠磷酸二氢（的NaH 2 PO 4）和42.3毫升，1M钠磷酸氢二钠（的NaH 2 PO 4）。调整pH值至7.0 注意：不要使用20％SDS，如果有降水。这是正常的乳白色悬浮液，当你添加SDS的解决方案。一旦你添加SDS的提取液置于60 ° C，以确保SDS是以及暂停。 </td></tr></table>

extraction of high molecular weight genomic dna from soils and sediments

土壤中微生物的基因组多样性和代谢创新是密切相关的陆地生态系统内的养分和能量流的庞大和相对未开发的的油藏。培养独立的环境基因组，也被称为宏基因组学，方针的前景前所未有的访问，这种遗传信息通路的重建和对高价值的治疗和生物转化过程中的功能筛选。然而，土壤中的微生物仍然是一个挑战，主要是由于在获得大量插入库生产质量足够高的分子量DNA的难度。在这里，我们介绍了从土壤和沉积物中提取的分子量高，微生物群落的基因组DNA的协议。分离出基因组DNA的质量是建设下游的测序和筛选应用环境大插入基因库的理想。 程序启动细胞裂解。微生物的细胞壁和膜机械（磨）和化学力（β-巯基乙醇）溶解。分离基因组DNA提取缓冲液，氯仿 - 异戊醇，异丙醇。受雇于裂解和提取步骤的缓冲区，包括异硫氰酸胍和十六烷基铵（CTAB），以保持超高分子量基因组DNA的完整性。根据您的下游应用，可进一步纯化分离出的基因组DNA，使用氯化铯（CSCL）梯度离心，从而减少杂质，包括腐殖酸。第一个程序，提取，大约需要8小时，不包括DNA定量的一步。 CsCl梯度超速离心法，是为期两天的过程。基因组DNA在整个过程中，应被视为轻轻地防止剪切，避免剧烈震荡，反复苛刻的吹打。

Watch this Scientific Journal Video about Extraction of High Molecular Weight Genomic DNA from Soils and Sediments at JoVE.com

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

一个隔离土壤微生物群落的超高分子量和高品质的基因组DNA的方法来描述。

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 ...

extraction-high-molecular-weight-genomic-dna-from-soils

Research

JoVE Journal

Biology

25.5K 次观看.  University of British Columbia - UBC. 一个隔离土壤微生物群落的超高分子量和高品质的基因组DNA的方法来描述。

视频: Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

DNA Extraction from 0.22 &amp;mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

从0.22微米Sterivex过滤器和氯化铯密度梯度离心法的DNA提取

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

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.

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 ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for 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 blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

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 ...

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.

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 ...

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.

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 ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

粘蛋白琼脂糖凝胶电泳：蛋白质印迹高分子量糖蛋白


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

改进的高通量DNA提取板加载方法，降低污染的可能性

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...