我们特别感谢赛斯达斯特提供RNAP和MFD蛋白表达载体。我们也感谢谢尔盖Borukhov GreB和张丹提供技术支持。这项工作是支持由来自（按付款当日价格计算）国家卫生研究院的资助，由玛丽 - Josee和亨利克拉维斯在洛克菲勒大学（RTP）的奖学金。

使用这种方法是在报告的研究<a href="http://www.sciencemag.org/cgi/content-nw/full/327/5965/590/F3">，科学327，590-592（2010）波默朗茨和奥唐奈</a> 。 1。大会暂停的RNA聚合酶伸长复杂如下： 500 nm的终浓度的大肠杆菌大肠杆菌 RNA聚合酶σ70，他是5纳米的生物素标记的3.6 kb的DNA模板含有100μL缓冲液（20毫米的Tris - CL（pH值7.5），MgCl 2的8毫米，0.5毫米EDTA，5 T7A1发起人的最终浓度混合MM数码地面电视，10％甘油）为10分钟，在37 ° C。 APU和40微米的GTP，ATP和CTP的100微米添加一个额外的10分钟在37 ° C 2。停止RNAP伸长复杂固定到链霉亲和素珠是执行如下： 200μL和素磁性包被的磁珠（Invitrogen公司），分别加入上述反应在室温为10分钟。 3。固定的停止RNAP伸长复杂的纯化步骤如下： 珠（从上面）洗净，用0.9缓冲液含0.75 M氯化钠，200微克/ ml肝素，和20μg/ ml的单链DNA（上清每个磁分离洗涤步骤后删除。）接下来，5倍珠洗2次，用0.9毫升缓冲液A 4。形成一个复制叉下游相对头对RNA聚合酶如下： 重悬于100μLNew England Biolabs公司缓冲区4和10个单位SAP我（New England Biolabs公司）珠（从上面）10分钟添加37 ° C。珠子用0.9毫升缓冲液A洗3次，然后重悬于50μL快速T4连接反应缓冲液（New England Biolabs公司）。 2μL快速T4连接酶（New England Biolabs公司）随着6纳米预退火分叉DNA终浓度在室温为10分钟（RP10，RP22，RP25）。珠洗3次，用0.9毫升缓冲液A 5。大会的复制体，并在复制叉的领先链合成的起始执行如下： DnaB（六聚体）的448 pmol珠150缓冲液孵育（20毫米的Tris - CL（pH值7.5），8 毫米氯化镁2，0.5毫米EDTA，5毫米的数码地面电视，10％甘油）30秒时23 ° C。 4.9 pmol *第三的波尔（波尔三，他减去β），15 pmolβ，ATP的2毫米，60微米dGTP和dATP的分别加入200μL的体积和进一步的5分钟，37℃孵育复制开始后，一起加入10微克SSB和24微米的dATP和dTTP [α- 32 P] dTTP的和[α- 32 P] dCTP（具体活动，3,000-5,000 CPM / pmol）终体积为250微升。反应终止后10分钟后，加入12μL500毫米EDTA。 6。的分离纯化和浓度的放射性标记的DNA产品如下： 珠（从上面），煮沸后终止反应，以消除珠DNA。删除任何剩余的DNA，其余珠，珠治疗的蛋白酶K，10 UL量为30分钟，在50 ° C。珠子，然后煮和磁分离上清液删除。合并后的上清液含有DNA纯化使用Qiagen公司PCR清理套件。纯净放射性标记的DNA产品，然后在碱性琼脂糖凝胶电泳分析。 *在头RNA聚合酶停止伸长复杂如上，但是，σ70的情况下复制被省略。 代表性的成果： 头部碰撞与复制体RNA聚合酶通常在两种产品的长度为2.5 kb和3.6 KB结果。 2.5 kb的产品，暂停从复制体RNA聚合酶和拖延后，碰撞与RNA聚合酶的叉长度的DNA。 3.6 kb的产品，要么不完整，入住的RNA聚合酶启动子或部分复制体的RNA聚合酶头通读全长度的DNA和结果。一个良好的或准确的结果表明，约50％的全长DNA产生。然而，在某些情况下会出现少占用由RNA聚合酶启动子和一个完整长度的DNA的比例更高，因为一个更大的replisomes人口是观察不遇到迎头的RNA聚合酶。复制的唯一完整长度的DNA（3.6 KB）在停止RNAP结果的情况下。 <img src="/files/ftp_upload/1919/1919fig1.jpg" alt="图1" /> 图。 1复制体是阻碍了头的RNA聚合酶 。 （一）实验装置。一个E。 COL我RNA聚合酶停止伸长复杂生物素标记的DNA包含如前所述T7A1发起人（9）组装。简单地说，一个停止伸长复杂所形成的孵化与生物素化DNA的RNA聚合酶全酶的APU，GTP，ATP和CTP此外还有10分钟的10分钟20 BPS下游发起人。链霉亲和素珠，添加额外的10分钟然后珠洗5次，每0.9毫升含0.75米，200 mg / ml的肝素，氯化钠和100 nm的单链DNA，以消除不稳定的RNA聚合酶- DNA复合物和多余的NTPS缓冲区。的DNA，然后与SAPI消化，水洗，结扎预退火分叉的DNA。珠被冲洗一遍之前的复制体大会。 （二）DnaB是那么的复制体组装和复制发起的存在（2巷）或缺乏（1巷）发起人是位于2.5 kb和前叉是面向对叉的直接转录。 RNA聚合酶。

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twin+DNA+pumps+of+a+hexameric+helicase+provide+power+to+simultaneously+melt+two+duplexes.">Twin DNA pumps of a hexameric helicase provide power to simultaneously melt two duplexes.</a> Cell. 109, 757-757 (2002).</li><li>Hanawalt, P. C., Spivak, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=coli+Transcription+repair+coupling+factor+(Mfd+protein)+rescues+arrested+complexes+by+promoting+forward+translocation.">coli Transcription repair coupling factor (Mfd protein) rescues arrested complexes by promoting forward translocation.</a> Nat Rev Mol Cell Biol. 9, 958-958 (2008).</li><li>Hanawalt, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+DNA+repair:+two+decades+of+progress+and+surprises.">Transcription-coupled DNA repair: two decades of progress and surprises.</a> Science. 266, 1957-1957 (1994).</li><li>Selby, C. P., Sancar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription-coupled+repair+and+human+disease.">Transcription-coupled repair and human disease.</a> J Biol Chem. 270, 4882-4882 (1995).</li><li>Liu, B., Alberts, B. 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洗涤停止伸长复杂与高盐的约束珠是至关重要的，以消除任何不稳定的非特异性结合的RNA聚合酶分子。 RNA聚合酶具有极高的亲和力引物 - 模板类型的DNA结构。因此，有必要消除这种非特异性的RNA聚合酶- DNA复合物，以执行复制的引物 - 模板。集中的放射性标记的DNA产品使用Qiagen公司的PCR纯化试剂盒也很关键，以获得足够高的放射性的信号，可以在凝胶观察。

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<td>Dynabeads M-270</td>
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direct restart of a replication fork stalled by a head-on rna polymerase

体内研究表明，复制叉头转录复合物被逮捕到遇到。然而，命运的复制体和RNA聚合酶（RNAP）后，头部相撞是未知的。在这里，我们发现， 大肠杆菌碰撞后的大肠杆菌复制体摊位一个头转录复杂，而是倒塌，复制叉保持高度稳定和最终恢复后，取代了从DNA的RNA聚合酶的伸长率。我们还发现，转录修复偶联因子，MFD，促进直接重新启动的叉，下面的碰撞，促进RNA聚合酶的位移。这些研究结果表明复制设备的内在稳定性和新颖的作用在促进过去一个RNA聚合酶块复制的转录偶联修复途径。

Watch this Scientific Journal Video about Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase at JoVE.com

Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

复制体的命运碰撞与头部的RNA聚合酶（RNAP）是未知的。我们发现，碰撞后的头对RNA聚合酶的复制体摊位，但恢复后，取代了从DNA的RNA聚合酶的伸长。 MFD促进便利碰撞后的RNA聚合酶的位移复制重新启动。

复制叉停滞了迎头RNA聚合酶直接重新启动

In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA ...

direct-restart-replication-fork-stalled-head-on-rna

Research

JoVE Journal

Biology

13.5K 次观看.  Rockefeller University. 复制体的命运碰撞与头部的RNA聚合酶（RNAP）是未知的。我们发现，碰撞后的头对RNA聚合酶的复制体摊位，但恢复后，取代了从DNA的RNA聚合酶的伸长。 MFD促进便利碰撞后的RNA聚合酶的位移复制重新启动。

视频: Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

瞬时转染293T细胞复制缺陷型逆转录病毒生产

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

通过注射和电穿孔的斑马鱼Otocyst的MIF Morpholinos直接运抵影响内耳发展

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

从小鼠胰腺RNA提取：核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

一个定量聚合酶重组酶扩增法的发展与内部阳性对照

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell&#39;s replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.