理查德W.贝林格和大卫L灰，应用科学家，Thermo Scientific的NanoDrop产品提供技术援助和拍摄。

1。微量核酸定量使用NanoDrop 2000C分光光度计<ol><li>首先，微量分光光度计样品保留系统的上部和下部的光学表面清洁干净的去离子水的2至3μL，吹打到较低的光学表面。 </li><li>关闭的杠杆臂，确保上层基座与去离子水接触。抬起杠杆臂和一个清洁，干燥，无尘的实验室擦拭光学表面擦去。 </li><li>打开NanoDrop软件，并选择核酸应用程序。使用一个小批量，校准移液器1缓冲液配药到较低的光学表面，以执行一个空白的测量。下的力臂，并选择"空白"，在核酸应用。 </li><li>一旦空白测量完成后，两个光学表面与一个清洁，干燥，无尘的实验室擦拭干净。 </li><li>选择适当的常数要测量的样品。 <table border="1"><tbody><tr><td> 样品类型 </td><td> 选择选项 </td><td> 用于计算含量恒定 </td></tr><tr><td>双链DNA </td><td> DNA的50 </td><td> 50 </td></tr><tr><td>单链DNA </td><td> DNA的33 </td><td> 33 </td></tr><tr><td>核糖核酸</td><td> RNA - 40 </td><td> 40 </td></tr><tr><td>寡糖</td><td>自定义</td><td> 15-150 </td></tr></tbody></table> 表1:典型的核酸直接A280吸光度测量，使用一个NanoDrop微量分光光度计，一个传统的试管为基础的分光光度计使用一个TrayCell微蜂窝比色皿，在结合PicoGreen检测微量NanoDrop fluorospectrometer的浓度范围。 </li><li>免除到较低的光学基座1μL的核酸样品，并关闭力臂。由于测量是独立的体积，样品只需要，弥合两者之间的光学表面为测量的差距。 </li><li>在应用软件中，选择"测量"。该软件会自动计算出的核酸浓度和纯度比例。下面的示例测量，审查的光谱输出。 </li><li>该软件会自动计算出的核酸浓度和纯度比例。样品的测量，光谱图像，以评估样品的质量审查。 </li><li>一个典型的核酸样本，将有一个非常有特点的姿态。 <img src="/files/ftp_upload/2565/2565fig1.jpg" alt="图1" /> 图1一个典型的核酸样本将有一个非常有特点的姿态。 </li><li>与特定的核酸分离技术相关的常见污染物的来源包括苯酚/ Trizol法和列提取。在苯酚/ Trizol法提取的情况下，残留试剂的污染可能是由220至240 nm之间的异常光谱，以及在260〜280 nm区域的变化。相反，从列提取的残留胍可能导致附近230纳米的高峰期，在从230纳米到约240纳米的槽的转变。 <img src="/files/ftp_upload/2565/2565fig2.jpg" alt="图2" /> 图2样品B和C的高峰和低谷的转变相比，样品A说明如何污染物可以影响核酸样品的光谱。 </li><li>为了准确地评估样品的质量，260/280或二百三分之二百六十○比率应在与整体的光谱质量结合分析。纯核酸通常产量的1.8〜260/280的比例和DNA和RNA的2.0〜260/280的比例，分别。这个比率是依赖于用于空白和样品测量的缓冲pH值和离子强度。酸性溶液中会根据代表0.2-0.3的比例，而一个基本的解决方案将超过代表比例由0.2-0.3。显着不同纯度率可能表明存在蛋白质，酚或其他污染物，达到或接近280 nm处的强烈吸收。二百三十零分之二百六十零纯度比一个"纯"核酸一般在1.8-2.2范围内的值的第二项措施是DNA的纯度。纯度的比例明显低于预期值低可能表明所使用的隔离技术，可能需要进一步优化。 </li><li> NanoDrop分光光度计也可以用来量化的蛋白质，使用直接吸光度或蛋白质比色法。为了确保正确的列形成和重复性，2μL的样品应为蛋白质样品。请参考以下的朱庇特视频:微量法测定蛋白浓度使用NanoDrop <a href="http://jove.unicartagenaproxy.elogim.com/index/Details.stp?ID=1610">分光光度计</a> http://jove.unicartagenaproxy.elogim.com/index/Details.stp?ID=1610 </li></ol><ol><li>为了证明高灵敏度的微量核酸定量使用NanoDrop 3300，PicoGreen荧光试剂盒使用。首先，室温平衡试剂盒标准和所有核酸样品。荧光的样品对光敏感，应保存在琥珀或铝包裹管。 </li><li> PicoGreen工作将需要的解决方案，以及来衡量所有的标准和样品准备足够的1X TE缓冲液。准备无核酸水每制造商的协议所提供的20X TE缓冲液稀释1xTE缓冲区。反应体积的范围可以从200μLUL低至10。在这个例子中，10μL的反应体积会有所准备。 </li><li>准备在2X最终所需浓度无核酸小瓶或试管连续稀释的双链DNA标准。标有"无核酸琥珀色或金属箔覆盖的管个人转移到5μL每个稀释的dsDNA标准。 </li><li>分装5μL适当标记的核酸无琥珀色或金属箔覆盖管到每个感兴趣的双链DNA样本。 </li><li>根据制造商的协议，准备PicoGreen工作液。包含标准或利益的样品（5μL在这个例子中）转让PicoGreen工作液等体积每管。 </li><li>结合1X TE缓冲液和PicoGreen工作液等量准备阴性对照，作为对照品溶液。 </li><li>每个反应管中的内容进行彻底混合轻柔吹打，并在室温下孵育5分钟的反应。所有的标准品和样品相同的温度测量前应平衡，荧光信号受温度影响。 </li><li>为了准备仪器进行测量，先清洁的样品保留系统的下限和上限的表面。吸取2至3μL去离子水到较低的光学表面，然后关闭打开的杠杆臂，并用不起毛的干净无污点的上部和下部的底座，实验室擦拭。确保表面皮棉，然后再继续作为皮棉可以表现出荧光激励源的存在，可能会干扰测量。 </li><li>打开仪器软件，选择"核酸"应用程序，并选择"PicoGreen双链DNA"选项。 </li><li>空白的文书，加入2μL的1X TE较低的底座，关闭的手臂，并选择"空白"，在仪器软件领域。一旦完成空白，污点从样品保留系统两面空白的解决方案。 </li><li>轻柔吹打混合对照品溶液。使用低保留的提示，加入2微升到较低的底座和密切的手臂。 </li><li>选择测量类型中的"参考"，然后选择所需的单位。点击"测量"来启动测量周期。 </li><li>当测量周期完成后，打开手臂和彻底印迹样品保留系统表面。测量高达五复制参考，使用为每个复制的新鲜等份。 </li><li>额外的标准，建立标准曲线，重复的过程。可用于最多七个标准。该软件的设计能够存储多达五个，每个标准重复。重复的标准是平均生成一个自动确定从该样品浓度的标准曲线。 </li><li>标准曲线一旦完成后，选择"样本"标签，进入各自的样品编号信息，并测量每个未知样品。 </li></ol>

<ol>
	<li>Wilfinger, W. W., Mackey, K., Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+and+Ionic+Strength+on+the+Spectrophotometric+Assessment+of+Nucleic+Acid+Purity.">Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity.</a> BioTechniques. 22, 474-481 (1997).</li><li>Voolstra, C., Jungnickel, A., Borrmann, L., Kirchner, R., Huber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Quantification+of+Nucleic+Acids:+LabelGuard+enables+photometric+quantification+of+submicroliter+samples+using+a+standard+photometer.">Spectrophotometric Quantification of Nucleic Acids: LabelGuard enables photometric quantification of submicroliter samples using a standard photometer.</a> Implen Applications Note. , (2006).</li><li>Jr, I. n. g. l. e., D, J., Crouch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Spectrochemical analysis. , XV-590 (1988).</li><li>Van Lancker, M., Gheyssens, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+four+frequently+used+assays+for+quantitative+determination+of+DNA.">A comparison of four frequently used assays for quantitative determination of DNA.</a> Anal. Lett. 19, 615-623 (1986).</li></ol>

菲利普德斯贾丁斯和黛布拉康克林受雇于Thermo Scientific的NanoDrop产品，公司生产本文中使用的仪器。

这里介绍的定量协议是基于最广泛接受的微量系统。这种系统提供了实用的替代传统的核酸定量方法，依赖于更大的样品量和遏制仪器的使用。 NanoDrop微量技术采用了一种依赖对所测样品，形成液柱的表面张力特性的样品保留系统。重要的是，样品与适当的列形成上下光学测量表面接触。如果在任何点的测量结果似乎不准确或不重复，这是最有可能的样本异质性或液柱断裂的结果。 洗涤剂和异丙醇的清洁剂，因为它们可能uncondition基座测量表面不建议。一个无条件的基座已经失​​密时，会发生底座的疏水表面性质，导致在一个扁平化的样品液滴。在测量过程中，无条件的底座可以导致液柱断裂。 <img src="/files/ftp_upload/2565/2565fig3.jpg" alt="图3" /> 图3。无条件的表面特征是由水滴压扁。 Uncondtioned基座表面，可翻新使用NanoDrop立柱翻新剂（PR - 1，Thermo Scientific的NanoDrop产品）。一个翻新复合一层薄薄的上部和下部的底座表面，让其干燥〜30秒，然后使用一个清洁，干燥，无尘的实验室擦拭磨去。成功修复的状态是由水珠串适用于底部的基座表面时，液滴的特点。 <img src="/files/ftp_upload/2565/2565fig4.jpg" alt="图4" /> 图4:一个翻新的表面特征是由水滴钉珠。 微量定量系统，大大降低了样品消耗和更传统的量化系统相比显着增加的浓度范围。尽管微量定量甚至常常成为一个涉及有限的细胞团，如穿刺活检和激光捕获显微切割，效率和易于使用这种方法已被广泛接受的替代传统的核酸定量方法的情况下，使技术当样本是充足的。

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<td>NanoDrop 2000c</td>
<td>&nbsp;</td>
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<td>ND-2000C</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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nanodrop microvolume quantitation of nucleic acids

生物分子检测，不断地被开发使用的材料逐步数额较小，往往排除了使用传统的试管为基础的手段，为那些可以进行微量定量的核酸定量。 NanoDrop微量样品保留系统（Thermo Scientific的NanoDrop产品）功能相结合光纤技术和自然表面张力特性，捕捉和保留微量的样品，独立于传统的遏制仪器，如试管或毛细血管。此外，该系统采用更短的路径长度，在一个广泛的核酸浓度测量的结果，从根本上消除了需要进行稀释。波谱分析所需的样本量的减少也有利于整个许多分子的工作流程纳入额外的质量控制步骤，提高效率，并最终导致下游的结果，以更大的信心。 有限质量高灵敏度荧光分析的需求也出现了与最近的实验进展。使用相同的微量样品保留技术，荧光测量可能与2μL材料，使荧光检测量的需求将大大减少。 10μL或更少的这种microreactions现在可以使用一个专用的微量fluorospectrometer。 两个微量核酸定量协议将表明，使用集成的采样系统作为替代传统的试管为基础的协议的实际保留。首先，直接的A260吸光度的方法，使用微量分光光度计描述。这是一个基于荧光的方法，使减少量的荧光反应，用微量fluorospectrometer示范。这些新技术使核酸的浓度从1 pg /μL的15,000纳克/μL样品消耗最小的评估。

Watch this Scientific Journal Video about NanoDrop Microvolume Quantitation of Nucleic Acids at JoVE.com

NanoDrop Microvolume Quantitation of Nucleic Acids

NanoDrop微量系统实用，高效的替代传统的核酸定量方法的使用是通过两个微量核酸定量协议的示范。

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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nanodrop-microvolume-quantitation-of-nucleic-acids

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

201.4K 次观看.  Thermo Scientific NanoDrop Products.  中文 Deutsch Français 

视频: NanoDrop Microvolume Quantitation of Nucleic Acids

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

科研

生物学

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

核酸结构分析及建设3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

多不饱和脂肪酸的膳食补充剂<em&gt;秀丽隐杆线虫</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

NanoDrop微量核酸的定量

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