这项研究的支持，美国能源部科学办公室（BER）的合作协定“没有。 DE - FC02 - 02ER63453，国家科学基金会的微生物基因组测序计划（奖号码0626826和0731916）。我们感谢他的技术专长和建议，并K.埃里克Wommack约翰玻璃，他与环境样品的采集援助。

1。制备解决方案在执行羟基磷灰石色谱之前，必须准备和磷酸盐缓冲液必须正确水合羟基磷灰石。 <ol><li> 1M磷酸溶液，pH为6.8：溶解在1L容量瓶119.98克磷酸二氢钠，在1L无菌DEPC 处理的H 2 O准备在1L容量瓶一个1M钠磷酸氢二钠溶液溶解在1L无菌DEPC处理的H 2 O磷酸钠二元141.96克在以1:1的比例，结合单和DI -解决方案。将烧瓶上不小的轰动板和混合使用磁力搅拌棒。调节pH值至6.8，加入氢氧化钠（增加pH值）或磷酸（以降低pH值）。 </li><li> 0.12M磷酸盐缓冲液：在500ml烧瓶中，结合2.5毫升，pH值6.8，10％的十二烷基硫酸钠（SDS），1M磷酸溶液，0.5M EDTA5毫升30毫升。添加无菌DEPC处理水的225毫升量。调节pH值至6.8。添加无菌DEPC处理水至250ml。高压灭菌。 </li><li> 0.18M磷酸盐缓冲液：在500ml烧瓶中，结合2.5毫升，pH值6.8，10％的十二烷基硫酸钠（SDS），1M磷酸溶液，0.5M EDTA5毫升45毫升。添加无菌DEPC处理的H 2 O的225毫升量。调节pH值至6.8。添加无菌DEPC处理的H 2 O至250ml 。高压灭菌。 </li><li> 020的磷酸盐缓冲液：在500ml烧瓶中，结合2.5毫升，pH值6.8，10％的十二烷基硫酸钠（SDS），1M磷酸溶液，0.5M EDTA5毫升50毫升。添加无菌DEPC处理的H 2 O的225毫升量。调节pH值至6.8。添加无菌DEPC处理的H 2 O至250ml 。高压灭菌。 </li><li> 0.40 M磷酸缓冲液：在500ml烧瓶中，100毫升2.5毫升，pH值6.8，10％的十二烷基硫酸钠（SDS），1M磷酸溶液，0.5M EDTA5毫升结合起来。添加无菌DEPC处理的H 2 O的225毫升量。调节pH值至6.8。添加无菌DEPC处理的H 2 O至250ml 。高压灭菌。 </li><li> “1.00M磷酸缓冲液”（实际浓度的磷酸盐，在这个解决方案是91万）：在500ml烧瓶中，结合30毫升1M磷酸溶液，pH6.8，10％的十二烷基硫酸钠（SDS），2.5毫升，5ml的0.5M EDTA。添加无菌DEPC处理水的225毫升量。调节pH值至6.8。添加无菌DEPC处理的H 2 O至250ml 。高压灭菌。 </li><li>羟基磷灰石水化：重出1克的羟基磷灰石，并添加至50ml管。 6毫升的0.12M磷酸盐缓冲液添加羟基磷灰石和颠倒混合。之前使用带来的温度为60℃，调匀。长时间储存​​在4 ° C </li><li>在使用之前，在60 ° C平衡所有的磷酸盐缓冲液和水合羟基磷灰石</li></ol> 2。经济柱的制备<ol><li>关闭活塞。与去离子H 2 O冲洗多次反相柱和调迁列多次。 </li><li>从经济型列删除活塞和高压灭菌消毒列。 </li><li>更换和关闭活塞。添加Sigmacote1毫升无菌的Econo列和大衣所有的玻璃表面。打开活塞和倒出。 </li><li>列循环水浴，设定温度为60 ° C。 einsure跨列列的包装，如铝箔或泡沫保温管，绝缘剂的热损失量最少的是建议。 </li><li>冲洗两次的Econo列无菌，无RNase H 2 O，然后两次无菌，无RNase 0.12M磷酸缓冲液。 </li></ol> 3。 Hydroxypaptite Chromotography <ol><li>确保活塞是封闭的，慢慢加入事先准备好的，悬浮羟基磷灰石2毫升的Econo柱底部使用无菌2毫升血清吸管。 </li><li>允许羟基磷灰石重力下为30分钟解决。 </li><li>排列，并关闭活塞的缓冲。 0.12M磷酸盐缓冲液核酸结合在一个终体积为500μL孵育在60 ° C时在加热块或水浴10分钟。 </li><li>快速申请准备在0.12M磷酸盐缓冲液中的羟基磷灰石（使用2毫升血清吸管，确保不打扰列）的核酸。 </li><li>允许核酸绑定到30分钟的羟基磷灰石。 </li><li>列下放置一个15毫升管，打开活塞，收集初始样本含有单链DNA。关闭活塞。 </li><li> 0.12M磷酸盐缓冲液添加6毫升一定不要去打扰列，打开活塞，并在上一步15毫升管收集的单链DNA的羟基磷灰石。关闭活塞。 </li><li>加入1体积的苯酚：氯仿：异戊醇（25:25:1，V / V / V）溶液管，混合大力离心15分钟，并在3500 XG。上清含有单链DNA转移到一个新的15毫升管。 </li><li>重复使用020磷酸盐缓冲至E6毫升3.76和3.87琵琶和列净化RNA一定要使用一个新的15毫升管收集。 </li><li>使用6毫升的0.40M磷酸盐缓冲液洗脱和净化一定要使用一个新的15毫升管收集的列的双链DNA重复3.76和3.87。 </li><li>重复使用1.00M磷酸盐缓冲地带的任何残余的双链DNA一定要使用一个新的15毫升管收集列的6毫升3.76和3.87。 </li></ol> 4。脱盐核酸酸样品<ol><li>传输样品45毫升一个Amicon超4离心过滤设备与装备切断Ultracel膜的重量为30,000分子。 </li><li>集中样品&lt;500μL纺纱6000 XG，30℃，5分钟（或直到集中量达到）在一个固定角度转子。丢弃流量通过。 </li><li>其余样品（S）的Amicon Ultra - 4的离心式过滤装置，并带来了量高达45毫升用RNase免费1X TE缓冲液。 </li><li>集中样品&lt;500μL纺纱6000 XG，30℃，5分钟（或直到集中量达到）在一个固定角度转子。丢弃流量通过。 </li><li> 45毫升的RNase免费1X TE缓冲液添加的Amicon Ultra - 4的离心过滤设备。 6000 XG，30离心浓缩样品&lt;500μL℃，5分钟（或直到集中量达到）在一个固定角度转子。丢弃流量通过。 </li><li>重复步骤4.5一个额外的5倍，完全淡化核酸样品（S）。 </li><li>将剩余的样本（S）1.7毫升无菌Eppendorf管。新增的十分之一体积3M醋酸钠，pH值7.0，两卷100％的乙醇，Glycoblue加入1μl。由涡旋混合。 </li><li> 28000 XG，4℃，60分钟离心。倒出作出一定不要去打扰颗粒。 70％的乙醇添加300μL。自旋为28,000 XG，室温为10分钟。倒出，干燥，​​并在适当体积的无RNase的TE缓冲液重悬每个分数。 </li></ol> 5。代表性的成果： 图1总结了从混合病毒组合使用羟基磷灰石色谱分馏的单链DNA，双链DNA和RNA的核酸提出的方法。这种方法是利用电荷相互作用之间的核酸带负电荷的磷酸骨架和带正电的CA 2 +离子目前在羟基磷灰石和核酸亚型的有效分离（单链DNA，双链DNA和RNA），允许用磷酸盐缓冲液浓度增加12。 在体外的“社区”已知的单链DNA（M13mp18），双链DNA（λ）和RNA的羟基磷灰石的分离（MS2和phi6）病毒的基因组如图2所示。核酸结合在平等的浓度，适用于羟基磷灰石柱和洗脱使用的磷酸盐缓冲液浓度的增加。每个核酸亚型洗脱独立于其他与低于9％，从一小部分结转下。 这项技术的应用从切萨皮克湾收集到了病毒社会的孤立的核酸如图 3所示。羟基磷灰石柱分离纯化病毒总核酸产量。独立的单链DNA，RNA和双链DNA组成的基因材料与0.12M，020和0.40M/1.00M的磷酸盐缓冲液浓度分别洗脱。双链DNA洗脱0.40M和1.00M磷酸盐浓度，在这种情况下，占主导地位这一病毒的社区相比，单链DNA和RNA的基因型。这个观察是符合预期的海洋病毒群落组成和河口环境大多数可收回核酸双链13。 <img src="/files/ftp_upload/3146/3146fig1.jpg" alt="图1"/> 图1。核酸分馏的羟基磷灰石色谱法的流程图。在0.12M磷酸盐缓冲液制备的单链DNA，双链DNA和RNA的混合物加热至60 ° C，并应用到一个羟基磷灰石柱保持在恒定的60℃使用循环水洗澡。 （单链DNA，RNA和双链DNA）核酸是由羟基磷灰石洗脱与一个含有磷酸盐缓冲浓度的增加。由美国微生物学协会的许可，这个数字已被复制，最初是在安德鲁斯- Pfannkoch等功能。 2010年12。 <img src="/files/ftp_upload/3146/3146fig2.jpg" alt="图2"/> 图2。使用羟基磷灰石色谱已知的病毒核酸的分离。平等浓度M13mp18单链DNA，MS2的ssRNA，phi6双链RNA和lambda双链DNA（泳道2-5分别）相结合（6车道），并应用到羟基磷灰石柱。 SINGLE -双链DNA（M13mp18），RNA（MS2/phi6）和双链DNA（λ）分别独立使用0.12M（8车道），0.18M（9巷）和0.40M/1.00M（10巷）羟基​​磷灰石柱洗脱从。一个1KB的阶梯（1车道，7）是用来确定基因组大小。由美国微生物学协会的许可，这个数字已被复制，最初是在安德鲁斯- Pfannkoch等功能。 2010年12。 <img src="/files/ftp_upload/3146/3146fig3.jpg" alt="图3"/> 图3：从社会孤立的切萨皮克湾内的病毒核酸的分离。核酸分离并应用到羟基磷灰石柱。单链DNA（巷0.12M），RNA（020道）和双链DNA（车道0.40M/1.0M）独立使用0.12M，020万和0.40M/1.00M的磷酸盐缓冲液浓度，分别洗脱。您好地下DNA梯状条带（巷L1）和1KB梯（车道二级），用于直观地确认近似分子量和核酸的质量。由美国微生物学协会的许可，这个数字已被复制，最初是在安德鲁斯- Pfannkoch等功能。 2010年12。

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没有利益冲突的声明。

羟基磷灰石色谱这里介绍的方法是核酸病毒混合的组合，当目标是研究社会的总核酸组成的分馏的高效和强大的工具。一般来说，单链DNA，RNA和双链DNA列PHO磷酸盐缓冲液浓度比约〜0and 0.40M分别洗脱。然而，每一个羟基磷灰石的制备可以有一个稍微不同的组成和流出曲线，所以重要的是测试每个准备使用已知的核酸（例如，从耕地的病毒编写）。这将允许用户以确定具体含有磷酸盐缓冲液洗...

<table border="1"><tbody><tr><th>材料名称</th><th>公司</th><th>目录编号</th><th>目录编号</th></tr><tr><td>柱的Econo - </td><td> Bio - Rad公司</td><td> 737-0717 </td><td> 0.7厘米编号封装/ 2 </td></tr><tr><td>羟基磷灰石</td><td> Bio - Rad公司</td><td> 130-0520 </td><td> DNA的二级生物凝胶HTP凝胶，100g/td&gt; </tr><tr><td>磷酸钠，二氢</td><td> VWR </td><td> VW1497 - 01 </td><td>一水，水晶500克</td></tr><tr><td>磷酸三钠，磷酸氢二铵</td><td> VWR </td><td> VW1496 - 01 </td><td>无水，粉末500克</td></tr><tr><td> DEPC处理过的水</td><td> Invitrogen公司</td><td> AM9922 </td><td> 1L </td></tr><tr><td> 10％SDS溶液</td><td> Invitrogen公司</td><td> 24730-020 </td><td>超纯水，1L </td></tr><tr><td> 0.5M EDTA </td><td> Invitrogen公司</td><td> AM9262 </td><td> pH值8.0，1L </td></tr><tr><td> Sigmacote </td><td> Sigma - Aldrich公司</td><td> SL2 - 25ML </td><td></td></tr><tr><td> 2毫升血清pippette </td><td> VWR </td><td> 89130-884 </td><td>聚苯乙烯，无菌</td></tr><tr><td>屋宇署猎鹰离心管</td><td> VWR </td><td> 21008-936 </td><td> 15毫升，无菌</td></tr><tr><td>酚：氯仿：异戊醇（25:24:1，V / V / V） </td><td> Invitrogen公司</td><td> 15593-031 </td><td>超纯，100毫升</td></tr><tr><td> Amicon超4离心设备</td><td> Millipore公司</td><td> UFC803024 </td><td> Ultracel - 30膜</td></tr><tr><td> 20X TE缓冲液，核糖核酸酶自由</td><td> Invitrogen公司</td><td> T11493 </td><td> 100毫升</td></tr><tr><td> Glycoblue </td><td> Invitrogen公司</td><td> AM9516 </td><td> 15mg/ml </td></tr></tbody></table>

separation of single-stranded dna, double-stranded dna and rna from an environmental viral community using hydroxyapatite chromatography

病毒，尤其是噬菌体（噬菌体），是地球上 1,2数量最多的生物实体。病毒调节宿主细胞的丰富性和多样性，促进养分循环，改变宿主细胞的表型，影响宿主细胞和病毒通过基因的横向转移3社区的演变。大量的研究有突出的病毒和它们在各种自然环境的功能潜力惊人的遗传多样性。 宏基因组技术已被用于研究生物分类的多样性和复杂的病毒，其成员中含有单链DNA（单链DNA），双链DNA（dsDNA）的和RNA 的基因型4-9的组合功能的潜力。当前协议，用于研究环境的DNA含有或RNA含有病毒库的建设需要一个初始的核酸治疗，为了消除非定标模板10。然而，一个全面的了解病毒的社会和病毒的多样性集体的基因补充，需要所有成员的知识，不论基因组组成。分馏纯化核酸亚型提供了一个有效的机制来研究不牺牲社会的基因特征的一个子集的病毒组合。 羟基磷灰石，磷酸三钙的结晶形式，已受聘于核酸的分离，以及蛋白质和微生物，自1960年以来 11 。通过利用带正电荷的钙 2的电荷相互作用的羟基离子和带负电荷的核酸亚型的磷酸骨架，它是可以优先洗脱每个独立于其他的核酸亚型。我们最近采用这一战略的独立分馏，准备DNA 测序 12单链DNA，双链DNA和RNA含有病毒的基因组。在这里，我们提出了一种单链DNA，双链DNA和RNA病毒核酸病毒混合组合使用羟基磷灰石chromotography分馏和恢复的方法。

Watch this Scientific Journal Video about Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography at JoVE.com

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

我们描述了独立于环境病毒社区的单链DNA，双链DNA和RNA分子的有效方法。核酸是分次使用含有磷酸盐缓冲液浓度的增加羟基磷灰石色谱。这种方法允许所有病毒从环境样品中的核酸类型的隔离。

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

separation-single-stranded-dna-double-stranded-dna-rna-from-an

Research

JoVE Journal

Immunology and Infection

31.4K 次观看.  The J. Craig Venter Institute. 我们描述了独立于环境病毒社区的单链DNA，双链DNA和RNA分子的有效方法。核酸是分次使用含有磷酸盐缓冲液浓度的增加羟基磷灰石色谱。这种方法允许所有病毒从环境样品中的核酸类型的隔离。

视频: Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

通过“Acufection”和它的皮肤研究中的应用的经济型DNA递送系统的开发

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.