我们感谢塞缪尔谢泼德，彼得Bazeley，约翰大卫贝尔基因MRI网页管理。这项工作是由美国国家科学基金会职业奖“，内含子细胞的作用研究”[授予MCB - 0643542]。

纸中使用的所有节目已经使用perl编写的，并已经使用PHP创建的所有网页。 1。起点： 打开http://mco321125.meduohio.edu/〜jbechtel / gmri /首页的在线基因组的MRI包。 Web资源，还提供了“帮助（How-to/README）”链接，而对基因组的 MRI和类似的算法所有公布的材料，在“链接到相关的资源”列出的链接方案的说明/解释。 2。输入序列（S）的制备和上传。 创建一个与FASTA格式的序列（S）文件，启动一个GMRI分析会话。这种格式中的每个核苷酸序列之前，应先用一个“&gt;”字符表示一个标识符，同一行的简短描述了这个序列，起跑线。核苷酸序列GMRI分析还允许字符R，Y，N，X等Hwever，非A，T，C，G字符不会被处理程序，将被跳过。重复的元素，其中有被“蒙面”（“N”代替）的序列可以作为输入。请注意，序列字符大小写不敏感的。 <ol><li>点击基因MRI主页上的“ 启动或恢复 ”按钮开始一个GMRI会议。这需要用户到一个地方的核苷酸序列可以上传的页面。 </li><li>复制并粘贴您的FASTA格式的序列或上传文件，其中包含从您的本地计算机上使用的“选择文件 ”按钮的序列。 </li><li>点击“ 启动新的会话与此文件 ”按钮。应该会出现一个确认消息声明说：“ 你的序列已成功上传 ”，你也应该得到一个字母“GMRI标识符”[网站调用它一个“会话标签”为您的会话（例如b16yMj），上面的输入窗口可用于检索，并继续为会议后第一次使用长达两个星期。 </li></ol>注：今后，输入序列简称为“userfile的”。 3。得到一个输入序列（可选）寡核苷酸的频率分布。 为了得到整个输入序列的寡核苷酸频率分布点击“SRI的分析仪”选项卡（上排） 。 SRI的缩写代表的短距离的不均匀性。此时，用户可以指定最高的寡核苷酸长度（从2到9个核苷酸，默认为6 NTS）的频率将被计算。这个选择是通过单击所需的选项内“ 最大的低聚物的大小”列表框。然后按“ 分析文件 ”按钮，启动计算。一个输入序列组成的粗糙表示将立即在本网页中显示为一个短表和“userfile.comp.tbl”下载。此表只有最和最丰富的寡核苷酸在输入序列。 所有可能的寡核苷酸的整个频率表生成名为“userfile.comp”的文件，它可以通过“下载的组成文件“链接获得。 注：SRI的分析仪计数的所有重叠的寡核苷酸的整套。 4。生成相同的输入序列（可选）寡核苷酸组成的随机序列。 （这个任务需要完成该协议的第3步）。 <ol><li> “SRI的发电机 ”的标签（上排），点击打开一个新网页，创建随机序列。选择使用本网页上的列表框中要生成的随机序列的样本数量。这些示例文件都将包含在“userfile的 ”输入序列相同数量和长度的随机序列。此外，如果一个输入序列中包含非A，T，C或G字符，随机序列将在相同的位置输入序列中的完全一样“N”的。 </li><li>选择的频率将被近似随机序列的寡核苷酸的长度最长。这可以通过检查所需的低聚物水平表（例如“4个碱基 ”四基寡核苷酸）在屏幕中心的单选按钮选择。这是必须指出的是，不仅在选择了低聚物级别的近似频率，而且较短的低聚物水平相应的频率，在输入序列，随机序列将包括。在寡核苷酸的小波动输入和随机序列的频率可能由于马尔可夫模型的随机序列的生成应用程序。 </li><li>通过点击“ 生成文件 ”按钮，启动该程序。如果输入序列是​​大的，它可能需要一两分钟，生成的随机序列。因此，用户应该等待，直到蓝色的“下载”链接在本页面底部出现。随机集被放置在文件名 ​​称，如“userfile.randX_Y”，其中 X是随机设置的数量和Y是选择低聚物水平（ 如 “userfile_rand2_4” ）。 </li></ol> 5。中档不均匀性（MRI）的输入和随机序列分析。 <ol><li>点击“ 磁共振成像仪 ”选项卡（上排），开辟了一个新的网页，分析中档的核苷酸序列组成的不均匀性。 </li><li>从分析“ 文件分析 ”列表框中（可在这里输入序列生成的随机序列集之间的选择），选择一个序列。 </li><li>要通过提供的清单中中分析， 选择的内容类型的MRI 。 （七内容选项可供选择：G + G + A，G + T;一个，G，C;或T） </li><li>选择窗口，内容丰富，内容贫乏的序列将通过“窗口大小”列表框（默认为50个核苷酸的有效范围是从30日至1000）的研究的长度。 </li><li>选择上限内容丰富，内容贫乏的地区， 门槛较低 ，分别。这些阈值可以在当前窗口中的特定核苷酸的确切数字（使用号码选项，在列表框中）或由这些核苷酸的百分比（使用百分比选项）中定义的窗口</li><li>毕竟五个选择已作出（例如：序列=“userfile的 ”;内容= 气相色谱 ;窗口大小= 50;的上限阈值= 35，降低门槛= 15），按分析文件 “按钮调用该程序。该方案通过从所选择的输入的所有序列连续扫描。在每一个步骤，它获得了当前序列的长度等于指定的窗口大小的段，计算核苷酸所选择的内容的数量或百分比是否高于上限或低于下限阈值。如果窗口不匹配或者标准，选择下一个重叠窗口（一个核苷酸转移）是相同的分析。当一个窗口被发现的序列符合其中一个内容丰富，或穷人组成的门槛要求，程序的输出文件保存在此窗口中的序列，生成一个图形输出穗。在此之后，程序跳转到下一个非重叠的相邻的窗口和恢复扫描过程，直到达到序列的结尾。 </li><li>计划完成后，显示到输出文件中的链接（名称为“userfile_GC_50_35 .. 15”上面的例子），在网页中显示的图形表示的结果是（见图1 ）。这个图形显示， 从userfile的所有输入序列连接成一个字符串，并为X轴水平黑线，在图所示的碱基长度（ KB）。沿输入序列的所有内容丰富的地区被标记为蓝色的“向上”尖峰，并为红色的“向下”尖峰的内容贫乏的地区。内容丰富，内容贫乏窗口总数显示在parenthses在此图的底部（分别为32和19）的传说。这个数字足以说明相对丰度和安排的MRI地区。同时，具体细节在输出文件中（见图3）。在这个文件中，所有的核苷酸序列片段，内容丰富或贫困标准和它们的坐标匹配，可作为沿输入文件列表中的用户，根据其连续职位。 </li><li>选择序列的MRI分析完成后，用户可以在相同的网页，通过改变参数和/或输入文件开始一个新的进程。例如，为了检查先前生成的随机抽样与相同的MRI参数＃1，用户只需要更改文件分析“选项， 并选择 ”userfile_rand1_4“文件， 然后再次按分析文件“按钮。一个新的文件和图形显示将取代旧的。所有检查结果，并根据每个“会话lable”（GMRI标识符）的数字将被保存和活动从过去的两个星期。为了节省的结果/图URES永久，用户应选择“ 下载文件 ”选项卡（上排）和下载整个会话或单个文件，根据需要。 </li><li>有了这个核磁共振成像仪的网页，用户可以研究<ul><li> （G + C） - 丰富和（A + T）丰富的地区</li><li>嘌呤（A + G）丰富和嘧啶（C + T）丰富的地区</li><li>丰富和氨基酸酮（G + T），（+）丰富的地区</li><li>一个丰富的和一个穷人的地区</li><li>富G -和G -贫乏地区</li><li>丰富的T -和T -贫困地区</li><li> C丰富和C -贫困地区</li></ul></li><li> 基因组MRI的最新版本有一个新的选择学习地区丰富的嘌呤（R）/嘧啶（Y）的交替模式，可能形成Z - DNA的构象。目前，这个选项是从Z - DNA的 “链接”，作为上述其他MRI地区相同的基础上。用户应选择的（RY + YR）的数量上限和下限，在扫描窗口重叠的核苷酸。该计划产生类似的图形化输出和交替嘌呤和嘧啶丰富和枯竭的DNA片段的文件。公认的Z - DNA的地区必须高度浓缩的交替R / Y基地（见审查F＆F 2011）。 </li></ol> 6。在基因组MRI包（可选）的附加 ​​程序。 基因组MRI资源也有非常具体的随机序列生成的两个高级选项。它们可通过“ 核磁共振发生器 ”和“ 的CDS发生器 ”第一行中的标签。 <ol><li> MRI发生器创建随机序列作为输入文件具有相同的寡核苷酸组成（ 斯里兰卡发生器类似）。不过，除此之外，随机序列模仿MRI一个特定的模式，由用户指定。在这个网页的用户应该从列表框中指定的一个特定的MRI模式来模仿。列表框包含所有已在本次会议审议通过核磁共振分析仪（如“userfile_GC_50_35 .. 15”）的模式。使用此选项生成一个随机序列，将选定的输入文件相同的寡核苷酸的组成和相同的GC丰富和扶贫模式，如“ userfile_GC_50_35 .. 15“。 </li><li> CDS的发电机是用于蛋白质编码序列的随机。它保留一个由用户指定的输入编码相同的氨基酸序列。此外，该方案保留在用户选择的输入表中指定相同的密码子和DI -密码子的偏见。 CDS的发电机的在线版本也可以接受一个输入蛋白质序列。只能通过提供独立的从主基因MRI网页下载的Perl脚本程序的所有其他选项。 </li></ol> 7。代表性的成果该协议允许用户研究核苷酸序列组成的不均匀。更重要的是，它也支持各种随机序列寡核苷酸逼近输入序列组成的一代。通常，复杂的真核生物的基因组序列是不均质的构成，而是代表了丰富的特定核苷酸序列片段的复杂镶嵌（例如，丰富的嘌呤，（G + T），丰富的，（A + T）丰富，等）。中档规模（30-1000 BP），这些模式是由蓝色峰值和较低的红色尖峰内容贫乏的分类（参见图1和2）显示所选内容丰富段的核磁共振分析仪的图形输出显示。通常情况下，在任何一个自然顺序（图1）内容丰富，内容贫乏的地区是时间顺序高于地区相应的随机序列的相同类型（图2）具有相同的寡核苷酸的数量组成。这些序列片段与中档核苷酸组成的不均匀性，可能是​​用户感兴趣的。他们是从基因组作进一步调查 MRI输出文件。 <img src="/files/ftp_upload/2663/2663fig1.jpg" alt="图1" /> 图1的核磁共振分析仪从步骤5.7图形输出的一个例子。上一个44人的内含子的样本已取得的结果。蓝色条代表沿着这些内含子富含GC的区域位置。红条代表的GC -差（或）MRI检查AT丰富的地区。 Y轴为给定的内容类型包含的上限和下限。 <img src="/files/ftp_upload/2663/2663fig2.jpg" alt="图2" /> 图2。 核磁共振分析仪输出的随机序列“userfile.rand1_4”。 该图形化CAL代表性的MRI使用SRI的发电机程序在一个随机生成的的序列。 <img src="/files/ftp_upload/2663/2663fig3.jpg" alt="图3" /> 图3：从核磁共振分析仪的文本输出文件的开头的一个例子。 全部内容丰富，内容贫乏的程序中检测到的序列是在过去的（第四位）列。在窗户的数量来衡量，它们的相对位置，显示在第一列。第二和第三列是内容丰富，内容贫乏的地区，分别指标。

<ol>
	<li>Bechtel, J. M., Wittenschlaeger, T., Dwyer, T., Song, J., Arunachalam, S., Ramakrishnan, S. K., Shepard, S., Fedorov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+mid-range+inhomogeneity+correlates+with+an+abundance+of+RNA+secondary+structures.">Genomic mid-range inhomogeneity correlates with an abundance of RNA secondary structures.</a> BMC Genomics. 9, 284-284 (2008).</li><li>Prakash, A., Shepard, S., Mileyeva-Biebesheimer, O., He, J., Hart, B., Chen, M., Amarachiniha, S., Bechtel, J., Fedorov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+forces+shaping+human+genomic+sequence+at+mid-range+scales.">Molecular forces shaping human genomic sequence at mid-range scales.</a> BMC Genomics. 10, 513-513 (2009).</li><li>Fedorov, A., Fedorova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3:+An+Intricate+Mosaic+of+Genomic+Patterns+at+Mid-range+Scale.">Chapter 3: An Intricate Mosaic of Genomic Patterns at Mid-range Scale.</a> Advances in Genomic Sequence Analysis and Pattern Discovery. , 65-91 (2010).</li><li>Shepard, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4:+Binary-abstracted+Markov+models+and+their+application+to+sequence+classificatio.">Chapter 4: Binary-abstracted Markov models and their application to sequence classificatio.</a> The characterization and utilization of middle-range sequence patterns within human genome [dissertation]. , 57-157 (2010).</li></ol>

不均匀中档规模的核苷酸组成（30-1000核苷酸）的地区是在复杂的真核生物的基因组overabundant的，可以找到任何地方（间隔区，内含子，外显子的非翻译区，重复元素）。这些地区经常与异常DNA的构象。例如，purine-/pyrimidine-rich序列往往形成DNA triplexes（H - DNA）;交替嘌呤/嘧啶碱基序列与Z - DNA的构象相关的（G + C）丰富的地区表现出的结构异常乙DNA和可容易骨干乳沟;（A + T）丰富的地区可能会形成一个不寻常的的结构 - 一个DNA平仓元素等（2010年费奥多罗夫＆Fedorova审查）。一些这些中档的模式（如（G + T）丰富的地区）几乎没有调查，仍有待深入探索和认同。我们的基因组的 MRI Web资源的主要目的是帮助用户为他们进一步的实验分析，并探索其可能的功能磁共振成像地区的鉴定。 MRI的地区的知识可以被纳入，并提高新一代基因预测程序（谢泼德2010年）和推进我们了解基因组的功能和属性。

<ul><li>计算机与互联网</li><li>核苷酸序列的文件进行检查。 </li></ul>

genomic mri - a public resource for studying sequence patterns within genomic dna

非编码基因组区域，在复杂的真核生物，包括间隔区，内含子，外显子的未翻译的部分，正在深刻地在他们的核苷酸组成的非随机的序列模式的复杂镶嵌组成。这些模式包括中档所谓的不均匀性（MRI）地区 - 序列30-10000的长度是由一个特定的基地或基地相结合，丰富（如（G + T）丰富，富含嘌呤等核苷酸）。与MRI地区是不寻常的（非B型）的DNA结构，往往是参与调控基因表达，重组，以及其他遗传过程（2010年Fedorova和费奥多罗夫）。一个坚强的内固定在核磁共振地区对突变，往往会减少他们的序列不均匀性，同时还支持这些基因组序列的功能和重要性（普拉卡什等人，2009年）的偏见的存在。 在这里，我们展示了一个可以自由使用互联网资源 - 基因组MRI程序包 - （柏克德等2008）基因组序列的计算分析，以便发现和描述在其中的各种磁共振成像模式。此包还允许使用的各种属性和通信水平的自然输入DNA序列的随机序列生成。此资源的主要目标是为了方便广大地区的非编码DNA仍然很少的调查，并等待深入探索和认同的检查。

Watch this Scientific Journal Video about Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA at JoVE.com

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

我们目前的基因组序列的分析计算公共网站。它可以检测出各种非随机核苷酸组成的DNA序列模式。这种资源也产生不同层次的复杂的随机序列。

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 ...

genomic-mri-public-resource-for-studying-sequence-patterns-within

Research

JoVE Journal

Biology

10.1K 次观看.  University of Toledo Health Science Campus. 我们目前的基因组序列的分析计算公共网站。它可以检测出各种非随机核苷酸组成的DNA序列模式。这种资源也产生不同层次的复杂的随机序列。

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

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

科研

生物学

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

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.

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 ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

研究年龄相关的基因组不稳定性使用 S。酵母年代的寿命模型

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

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 ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.