The authors acknowledge Liyi Xu for providing MDA reagents and protocols. We also thank David Feldman and Navpreet Ranu for discussions relating to this work. This work was supported in part by a Burroughs Wellcome Career Award at the Scientific Interface to PCB.

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The Broad Institute may move to file a patent application that includes aspects of this work.

المقايسات الرقمية وطرق القوية التي تمكن الكشف عن خلايا نادرة والعد من فرد من جزيئات الحمض النووي. ومع ذلك، لا تزال لا تطبق فحوصات الرقمية بشكل روتيني في المختبرات التحليلية، ويرجع ذلك جزئيا إلى تكلفة المعدات المتخصصة المرتبطة بالطرق المتاحة تجاريا. نحن هنا وصف مقاي?...

المقايسات الرقمية للحمض النووي الكمي (PCR الرقمي) 1-4 وقاعدة النظام (التسلسل) والتي تؤثر بشدة علوم الحياة والطب. توفر فحوصات الرقمية الكمي من التهم الجزيئية على نطاق المطلق (وليس نسبة إلى عنصر تحكم)، وتوفير حساسية عالية، مما المقارنات السطحية عبر التجارب، وبشكل حاسم، وتمكن من بناء قواعد البيانات الكبيرة التي تحتوي على بيانات قابلة للمقارنة 5 (الجدول 1). على مدى السنوات ال 15 الماضية، برزت كامل الجينوم التضخيم (WGA) جنبا إلى جنب مع PCR كأداة العامة لتضخيم الحمض النووي. مثل PCR، WGA مفيد للتطبيقات التحليلية وإعدادي عن طريق تضخيم عناصر عينة دقيقة تصل إلى المستوى الذي يمكن الكشف بسهولة أو استخدامها لتحليلات لاحقة مثل قاعدة التسلسل. على عكس PCR، WGA ليست محددة لموضع DNA معين، بدلا مما يسمح التضخيم من جميع متواليات في العينة، بما في ذلك تسلسل غير معروفة.هذا الاختلاف الجوهري بين PCR وWGA يجعل الطرق مكملة لبعضها البعض، وتثير تحديات مختلفة في تطبيقها. ارتفاع العائد من ردود الفعل وغ 6 يمكن التضخيم الروتيني من الحمض النووي الجيني من الجزيئات واحدة 7، 8 خلايا واحدة وغيرها من العينات الكتلة الحيوية منخفضة 9 لتقدير أو مزيد من التحليل. التحديات الرئيسية المرتبطة الكيمياء WGA هي الحساسية المفرطة لالملوثات والتضخيم غير المتكافئ عبر جزيئات قالب الفردية 6. برز ومع ذلك، WGA تكتسب شعبية والتسلسل وحيدة الخلية مثل &quot;تطبيق القاتل&quot; التكنولوجيا WGA 10، وقالب الكمي من قبل WGA المهم في العديد من مجالات التطبيق 7. تم الأجهزة الرقمية لالكمي الحمض النووي الموصوفة سابقا في مجموعة متنوعة من محكم بصمام وعديم الصمامات الشكل ميكروفلويديكنهاية الخبر 11-14، بما في ذلك فحوصات القائم على قطرة 15،16 (الجدول 2). ومع ذلك، نظم ميكروفلويديك التجارية للتحليل الرقمي تتطلب معدات متخصصة لإعداد رد فعل وكشف المنتج 17. على microfluidics المزودة بصمامات مخصصة مرنة، ولكنها تتطلب دقة التصنيع الدقيق وأنظمة التحكم بالهواء المضغوط 18. في حين أنها بسيطة نسبيا لجعل monodisperse قطرات متناهية الصغر، التى تستند إلى مستحلب المقايسات الرقمية 19،20، قراءات رقمية هي مرهقة من الناحية الفنية، والتي تتطلب إما على نطاق واسع التصوير واسعة المجال (على غرار تقنيات التسلسل الجيل المقبل من شعبية) 21،22 أو القائم على تدفق عالية السرعة كشف قطرة 23-25. من الناحية المثالية، فإن فحص الرقمية تكون بسيطة من انشاء لقراءات، والحد من الحاجة إلى أجهزة معقدة والسماح أعداد كبيرة من العينات في أن تقرأ بسرعة. نحن هنا تصف طريقة مبسطة لقراءات من المقايسات قطرة الرقمية التي تستخدم في الوقت الحقيقي التقليدي PCR طnstrument لقياس مضان الجزء الأكبر من المقايسات الرقمية القائمة على الحبرية. في حين أن نهج جديد يمكن تطبيقه على المقايسات PCR الرقمية، فإنه من المفيد بشكل خاص لفحوصات وغ الرقمية التي التناظرية المقايسات التضخيم في الوقت الحقيقي (التي تعطي نتائج ممتازة للPCR) هي إشكالية. يتم تطبيق WGA عادة عينات مع جزيئات قالب التي هي غير متجانسة في تسلسل، وطول، ومحتوى قاعدة. وتتضخم هذه الجزيئات قالب مختلف بمعدلات مختلفة 6، مما يستدعي استخدام إشارة (&quot;القياسية&quot;) عينة ذات خصائص مطابقة. في كثير من الأحيان، لا يوجد مثل هذا المعيار هو متاح، أو خصائص العينات الواردة غير معروفة. عدم تجانس المواد المدخلات والممتلكات التي تعتمد على طول كيمياء WGA 26 أيضا تعقيد تفسير النتائج من خلال خلق غموض في ما يجري كميا - كتلة المدخلات، وعدد المدخلات من الجزيئات، وهو مزيج من الاثنين، أو لا. Fومكونا، للتسلسل غير خصوصية تجعل الكمي متعددة التضخيم التشرد (MDA) أكثر حساسية للتلوث من الكمي PCR منذ الجزيئات الملوثة من أي تسلسل لديها القدرة على التدخل. وتتناول المقايسات الرقمية ميكروفلويديك التلوث خلال فصل جزيئات قالب والحد من كميات رد فعل مثل أن عددا أقل من الملوثات يتم أخذ عينات. هنا نستخدم طريقة WGA متحاور شعبية، MDA 27. وتجدر الإشارة، عدة كيمياء WGA الأخرى بما في ذلك PicoPlex وMALBAC 28 تعتمد بشكل حاسم على الخطوات النزوح متساوي الحرارة حبلا الأولية. خطوات متساوي الحرارة تؤدي إلى تفاقم التحدي المتمثل في تطبيق فحوصات في الوقت الحقيقي التناظرية لالكمي WGA. WGA لا يمكنها أن تمنع تماما أثناء الإعداد، مما يؤدي إلى متغير غير المرغوب فيها قبل التضخيم، ولا discretized (&quot;تدوير&quot;) بطريقة حيث يمكن أن تكون مدفوعة تكرار جزيئات غير متجانسة على الانتهاء، وتوقفت قبل الدورة القادمة مثل PCR 11 </suص&gt; (الشكل 1). صيغة الفحص الرقمي للWGA تتسع إجراءات الإعداد رد فعل نموذجية بسبب الفصل بين كل جزيء لتعداد عند نقطة الفحص (حتى ما قبل التضخيم لا يؤثر على النتائج) قراءات الأصلية تعني أن دقة مستقلة إلى حد كبير من الاختلاف في كفاءة التضخيم. الفحص يعتمد على قطرات إنتاجها في النفط مع وحدات التخزين موحدة، حيث أن مستوى الإشارة في قطرة عند نقطة الفحص سوف تعتمد على حجم القطيرات، ونحن لا نريد اتساق النتائج تعتمد على المتوسط ​​عبر توزيع أحجام قطرات. جعل قطرات monodisperse الآن إجراء القياسي (وقد نشرت حول 3500 أوراق قطرة monodisperse منذ 2013)، ولكنها تتطلب الأجهزة ميكروفلويديك 25. في الواقع، أكثر من ست شركات تطوير المنتجات التجارية المستقلة التي تعتمد على إنتاج مثل هذه القطرات، وصنع قطرات رقائق ميكروفلويديك هممتوفرة تجاريا 23،29. لهذه الدراسة، كنا أجهزة ميكروفلويديك مخصصة أنتجت في المنزل (انظر البروتوكول). مضخات حقنة لدفع التدفق من خلال الأجهزة وتتوفر أيضا تجاريا، ولكن بدلا من ذلك، يمكن أن تكون بديلا مع الحقنة وحيدة لتدفق يحركها الفراغ لخفض التكاليف 30.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Evagreen Dye</td>
			<td>Biotium</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>New England Biotechnologies</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phi29 DNA Polymerase Reaction Buffer</td>
			<td>New England Biotechnologies</td>
			<td>B0269S</td>
			<td>Vortex periodically until aggregate dissolves</td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>New England Biotechnologies</td>
			<td>N3011S</td>
			<td>Heated to 50C and quenched on ice prior to dilution</td>
		</tr>
		<tr>
			<td>Randomized MDA oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#39;-NNNNN*N-3&#39;</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#8217;-CGGCAAACGGGAATGAAACGCC-3&#8217; and 5&#8217;-TGCGGCAAAGACAGCAACGG-3&#8217;</td>
		</tr>
		<tr>
			<td>UltraPure Nuclease-free water</td>
			<td>Life Technologies</td>
			<td>10977-015</td>
			<td>UV-treated for 30m in Stratalinker 2400 before use (optional)</td>
		</tr>
		<tr>
			<td>ROX reference dye</td>
			<td>Life Technologies</td>
			<td>12223-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR optical strip caps</td>
			<td>Life Technologies</td>
			<td>4323032</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Agilent Technologies</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP (25uM each)</td>
			<td>Agilent Technologies</td>
			<td>200415</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler - Mx3000P qPCR system</td>
			<td>Agilent Technologies</td>
			<td>401403</td>
			<td></td>
		</tr>
		<tr>
			<td>Barrier pipette tips</td>
			<td>VWR</td>
			<td>89003-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-rad oil for evagreen</td>
			<td>Bio-rad</td>
			<td>186-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>JumpStart Taq ReadyMix</td>
			<td>Sigma-Aldrich</td>
			<td>P2893-100RXN</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple bulk readout of digital nucleic acid quantification assays

المقايسات الرقمية وطرق القوية التي تمكن الكشف عن خلايا نادرة وفرز الفردية جزيئات الحمض النووي. ومع ذلك، المقايسات الرقمية لا تزال لم تطبق بشكل روتيني، وذلك بسبب التكلفة ومعدات معينة المرتبطة بالطرق المتاحة تجاريا. هنا نقدم طريقة مبسطة لقراءات من المقايسات قطرة الرقمية باستخدام الوقت الحقيقي PCR التقليدية أداة لقياس مضان الجزء الأكبر من المقايسات الرقمية القائمة على الحبرية. نحن تميز أداء فحص قراءات بالجملة باستخدام خليط من قطرة الاصطناعية وقطرات الرقمي المتعدد التضخيم التشرد (MDA) فحص. الكمية MDA ولا سيما الفوائد من تنسيق رد فعل الرقمي، ولكن ينطبق أسلوب جديد لدينا على أي فحص الرقمي. لإنشاء بروتوكولات الفحص الرقمية مثل PCR الرقمي، ويقدم طريقة لتسريع وتبسيط فحص قراءات. لدينا منهجية قراءات الأكبر يجلب مزايا partitioneد فحوصات دون الحاجة إلى متخصص قراءات أجهزة القياس. يتم تخفيض القيود الرئيسية للمنهجية قراءات السائبة النطاق الديناميكي مقارنة مع منصات القطيرات العد والحاجة لعينة القياسية، على الرغم من أن متطلبات هذا المعيار هي أقل تطلبا من لتجربة في الوقت الحقيقي التقليدية. يستخدم الكمي كامل الجينوم التضخيم (WGA) لاختبار الملوثات في ردود الفعل وغ وهي الطريقة الأكثر حساسية للكشف عن وجود شظايا الحمض النووي مع تسلسل غير معروفة، مما يتيح أسلوب الوعد الكبير في مجالات التطبيق المختلفة بما في ذلك مراقبة الجودة الدوائية والبيولوجيا الفلكية.

في حين قراءات الأكبر التقليدي / في الوقت الحقيقي يمكن أن تستخدم في كل PCR الكمي والمقايسات WGA الكمية (الشكل 1)، المقايسات الكمية رقمية توفر مزايا (الجدول 1). في طريقة وصفها، وتلا المقايسات الرقمية في شكل قطرات صغيرة مع قياس نقطة نهاية الجزء الأكبر بسيط <s...

Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

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.

قراءات السائبة بسيطة الرقمية نوكليك حمض الكمي فحوصات

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

simple-bulk-readout-of-digital-nucleic-acid-quantification-assays

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8.2K Views.  Broad Institute. 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. 

Video: Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop Microvolume الكميات من الأحماض النووية

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

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.

كهرباء خالية من الحمض النووي متسلسل وعزل بروتين

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.

تحليل وبناء هياكل الحمض النووي مع 3DNA

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

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZ البلمرة فحوصات: بروتوكولات بسيطة واعتبارات

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

التحضير والتصوير، والكمي لالسطحية الجرثومي على الحركة فحوصات

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

كفاءة نوكليك حمض استخراج و16S الريباسي جين تسلسل للبكتيريا الجماعة توصيف

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

عزل الترشيح من الأحماض النووية: وبسيطة وسريعة استخراج الحمض النووي الطريقة

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

مقايسة تنحية نازعة لاكتات — طريقة بسيطة وموثوق بها لتحديد النشاط تنحية أوتوفاجيك الأكبر في خلايا الثدييات

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

بلانكوري Scrunching كما قراءات سلوكية كمية لتحسس المحفزات الضارة

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

بروتوكولات تفاعل البوليميراز المتسلسل الرقمية لقطرات النسخ العكسي المكونة من خطوتين للكشف عن SARS-CoV-2 وقياسه

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...