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生。 </li><li> BD P100管离心15分钟，在室温下在2,500克。转移到微离心管上方的机械分离器收集的血浆。 </li><li> P100管存储，含有血液分离器下方的压实，在-80°C，直到你准备开始DNA转raction。 </li></ol> 2.1从血液P100管（P100_DNA） <ol><li> P100的管保存在-80°C后血浆提取。在3至4个月，检索P100的管中，压实的血液从冷冻和解冻，在37℃的水浴中5分钟（ 图1A）。删除任何残留的血浆分离坐在上面。加入2毫升17％的蔗糖溶液（梯度溶液内部测试-未发表的数据）以上P100管分离（ 图1B）。离心管在室温下搅拌20分钟，在2,500克;这个过程中推压机械分离器管的顶部，产生的血沉棕黄层（ 图1C）的解决方案，包括三层。手动提取使用steriled钩状金属回形针（ 图1D）分隔。 </li><li>检索从每个管中（ 图1D）的机械分离器后，取出和抛弃它d为顶蔗糖层。转移到无菌的15毫升锥形管的中间层，代表的血沉棕黄层（BC）层。加入磷酸盐缓冲液（PBS），血沉棕黄层体积增加至3ml。按照制造商的建议，使用试剂从Qiagen公司Puregene的血包从白膜中提取的DNA除外的2,500克（而不是2000克）的离心速度。 </li><li>以溶解和去除红血细胞（RBC），加入9毫升红细胞裂解3毫升的血沉棕黄层。每个管倒置几次，并在室温下孵育10分钟，偶尔在温育过程中反相。向下旋转每种混合物在2,500克5分钟，弃上清。治疗其它颗粒在每个管中，用3毫升的细胞裂解缓冲液和旋涡，持续15秒。治疗的溶胞产物的蛋白沉淀溶液，用1毫升，在15秒的涡流。 </li><li> 2,500 g下离心5分钟，将上清转移到一个新的15毫升锥形管中3毫升100％的异丙醇。反转的新管10倍和离心机在2,500 G为3分钟。弃上清，加入1毫升70％的乙醇。倒置的锥形管打跑了几次，洗DNA沉淀。离心1分钟2500克。允许沉淀3分钟（颠倒放置管），在室温下干燥，然后暂停的的水化溶液用250ml的DNA在37℃下10分钟，并转移至预先标记的1.7毫升的微管。存储管在-80℃直到使用。 </li></ol> 2.2从血液的EDTA Ttube（EDTA_DNA） <ol><li>喷雾涂有10.8毫克K 2 EDTA，并储存在-80℃下，全血被收集在用于常规免疫血液学测试塑料vaccutainer管由于显像管不包含机械分离器，提取DNA不包括涉及机械分离器（2.1.1和2.1.2）的步骤。 </li><li>在3至4个月的储血，DNA被提取使用翠鸟的Flex（赛默飞世尔科技，沃尔瑟姆，MA，USA）。这是基于磁性颗粒技术，由细胞裂解/ DNA结合，一些洗涤步骤和DNA洗脱的自动化DNA提取系统。 </li><li> DNA提取中使用的试剂盒是：翠鸟Flex 24的DNA血液试剂盒（Qiagen，德国）。 </li></ol> 3。 DNA数量与质量评价基因组DNA的数量，质量和完整性进行评估相结合的方法，包括picogreen，光谱仪和电泳。 <ol><li>的基因组DNA的浓度来确定的NanoDrop和picogreen定量。 </li><li> Nanodrop分光光度计上测量从260/280的比例来确定的纯度。 </li><li>样品（500毫微克）还装载在1％琼脂糖凝胶中的DNA的完整性进行评估（退化）。 </li></ol> 4。 Genotypin克分析和质量控制<ol><li>五P100_DNA其相应的EDTA_DNA样本是随机选择使用定制的免疫芯片（DNA分析BeadChip芯片免疫）作进一步的分析。免疫芯片的Infinium含196,524多态性基因分型芯片是专为免疫遗传学研究5。 </li><li>对于每个样品，200 ng的DNA被扩增，分散，沉淀和重新悬浮在适当的杂交缓冲液中。 </li><li>免疫芯片上杂交变性的样品在48℃下至少16小时。 </li><li>杂交后，免疫芯片单碱基延伸反应和彩色处理。 </li><li>免疫芯片，然后成像利用Illumina Hiscan的磁珠阵列阅读器和归一化的钢丝强度的数据加载到Illumina公司GenomeStudio软件转换成基因型。基因型使用GenomeStudio自动调用算法被称为。质量控制volves的呼叫率&lt;95％的单核苷酸多态性（SNPs）的排除。 </li><li>的SNP的呼叫率是用来作为衡量免疫芯片检测的效率。算方法，是检测在芯片上（196524个SNP位点编码的，每个芯片上），达到足够的信号强度和质量，接收一个自动化的基因型分配的总数的百分比。 HapMap的对照DNA免疫芯片检测包括在至少相同的呼叫率预计能实现高品质的DNA（260/280比率为1.8或更高，和不存在下的降解）。质量控制涉及排斥的SNPs与呼叫率&lt;95％，在每个数据集。 </li></ol>

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没有利益冲突的声明。

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 ...

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

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JoVE Journal

Biology

41.9K 次观看.  Emory University School of Medicine and Children's Health Care of Atlanta. 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。

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

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

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.

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

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 ...

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.

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

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 ...

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 ...

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;

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

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.

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

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.

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

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 ...