لعينات ل arabidopsis والمعنية الاشعال PCR نشكر الأستاذ جاكو Kangasjärvi وجماعته، والإجهاد مجموعة نباتية، بيولوجيا النبات، قسم العلوم البيولوجية، جامعة هلسنكي. التجارب كانت تجرى مع الطريقة التقليدية السيدة Airi Lamminmäki. وقدمت عينات الماوس المعدلة وراثيا من قبل الدكتورة جانا Vesterinen، معهد الطب الحيوي / الكيمياء الحيوية، جامعة هلسنكي. هاريس أحادي النواة وقطع بساط هاريس هي علامات تجارية لشركة الاتصالات Shunderson جميع العلامات التجارية الأخرى هي ملك لشركة فيشر العلمية الحرارية والشركات التابعة لها.

<ol>
	<li>Doyle, J. J., Doyle, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+DNA+isolation+procedure+for+small+quantities+of+fresh+leaf+tissue.">A rapid DNA isolation procedure for small quantities of fresh leaf tissue.</a> Phytochemical Bulletin. 19, 11-15 (1987).</li><li>Kim, C. S., Lee, C. H., Shin, J. S., Chung, Y. S., Hyung, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+isolation+of+high+quality+genomic+DNA+from+fruit+trees+and+conifers+using+PVP.">A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP.</a> Nucleic Acids Research. 25, 1085-1086 (1997).</li><li>Neff, M. M., Neff, J. D., Chory, J., Pepper, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dCAPS,+a+simple+technique+for+the+genetic+analysis+of+single+nucleotide+polymorphisms:+experimental+applications+in+Arabidopsis+thaliana+genetics.">dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics.</a> The Plant Journal. 14, 387-392 (1998).</li><li>Michaels, S. D., Amasino, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+method+for+detecting+single-nucleotide+changes+as+polymorphic+markers+by+PCR.">A robust method for detecting single-nucleotide changes as polymorphic markers by PCR.</a> The Plant Journal. 14, 381-385 (1998).</li><li>John, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+isolation+of+RNA+and+DNA+from+plants+containing+polyphenolics.">An efficient method for isolation of RNA and DNA from plants containing polyphenolics.</a> Nucleic Acids Research. 20, 2381-23 (1992).</li><li>Wang, Z., Storm, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+mouse+tails.">Extraction of DNA from mouse tails.</a> BioTechniques. 41, 410-412 (2006).</li><li>Malumbres, M., Mangues, R., Ferrer, N., Lu, S., Pellicer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+High+Molecular+Weight+DNA+for+Reliable+Genotyping+of+Mice.">Isolation of High Molecular Weight DNA for Reliable Genotyping of Mice.</a> BioTechniques. 22, 1114-1119 (1997).</li><li>Yang, Y., Kim, J. Y., Soh, M. S., Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+gene+amplification+from+Arabidopsis+Leaves+Using+AnyDirect+system.">A simple and rapid gene amplification from Arabidopsis Leaves Using AnyDirect system.</a> Journal of Biochemistry and Molecular Biology. 40, 444-447 (2007).</li><li>Sambrook, J., Fritsch, E., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory. , (1989).</li><li>Meldgaard, M., Bollen, P. J. A., Finsen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+method+for+sampling+and+extraction+of+mouse+DNA+for+PCR.">Non-invasive method for sampling and extraction of mouse DNA for PCR.</a> Laboratory Animals. 3, 413-441 (2004).</li></ol>

ويرعى الإنتاج وحرية الوصول إلى هذه المقالة عن طريق شركة فيشر الحرارية والعلم

النهج PCR المباشر أثبتت هنا لتضخيم PCR يسمح مباشرة من كميات صغيرة من عينات غير منقى، والحد من الوقت اللازم وتبسيط سير العمل لمحطة والتنميط الجيني الحيوانية (الشكل 7). أظهرت أيضا هنا هو استخدام بروتوكول التخفيف في تركيبة مع النهج PCR المباشر (الشكل 3). ويوصى بروتوكول التخفيف مع عينات من الصعب (على سبيل المثال أوراق عمر النبات، والأنواع النباتية التي تحتوي على مركبات التدخل) أو مع amplicons (GC أو الغنية) لفترة طويلة. هذا البروتوكول هو مفيدة بشكل خاص عند بدء التجربة الجديدة PCR مباشرة، مما يسمح لتعظيم الاستفادة من رد الفعل. يمكن للبروتوكول أن تكون بمثابة أداة لمعالجة الصعوبات في بعض الأحيان مثل إنتاجية منخفضة المنتج بسبب الاختلافات في المواد الأنسجة و / أو الاشعال المستخدمة. وعلاوة على ذلك، عند تشغيل ردود فعل متعددة من نفس العينة، واستخدام بروتوكول يضمن التخفيف لا تستهلك كامل العينة في واحدةرد الفعل. وكانت الاتفاقية من أجل تنقية DNA من النباتات لPCR، لأداء تحلل الخلايا تليها عزل الحمض النووي باستخدام المكونات المختلفة التي يمكن أن تتداخل مع تحليل PCR يحتمل 8. لمحطات خاصة مع تحدي محتوى الفينول عالية، إضافة polyvinylpyrrolidone كان يستخدم تقليديا لإزالة المركبات التي ربط DNA بعد تحلل الخلايا 2،5. كما هو واضح هنا، يمكن تجنب هذه معقدة وتستغرق وقتا مكثفة الخطوات من خلال استخدام النباتات Phire كيت PCR المباشرة للكشف عن الحمض النووي المستهدف. ويمكن إهماله تحلل الخلايا والخطوات عزل الحمض النووي باستخدام تقنيات dCAPS الحساسة مباشرة من أوراق نبتة Arabidopsis (الشكل 2). كما هو موضح هنا، وبروتوكول التخفيف تدير بشكل فعال وجود عناصر إشكالية التي يمكن أن تتداخل مع PCR (الشكل 3). تقليديا، كان شرطا أساسيا لالتنميط الجيني الحيواني isolatiعلى من الحمض النووي الجيني من أنسجة الحيوانات السامة من خلال عملية تستغرق وقتا طويلا، تتميز الحضانة مطولة مع K بروتين الجلد لهضم والنسيج الضام، تليها الاستخلاص بالمذيبات العضوية، وهطول الأمطار الكحول، والخطوات الطرد المركزي 6،9،10. كما أظهر هنا، ويتحقق تبسيط هذه العملية من خلال استخدام الأنسجة الحيوانية Phire كيت PCR مباشرة، مما يسمح التنميط الجيني الفئران المعدلة وراثيا دون تنقية DNA السابقة (الشكل 4). المثال موضح في هذه المقالة يمثل تحديا لا سيما فيما كان يستخدم مجموعة واحدة فقط التمهيدي لتضخيم اثنين من شظايا مع اختلاف الحجم الكبير. على الرغم من صعوبة البروتوكول الفطرية، واستخدام نهج PCR المباشر بالتزامن مع بروتوكول تخفيف بسيط أدى إلى عوائد عالية من المنتجات PCR اثنين (الشكل 4). PCR المستندة إلى الكشف عن الحمض النووي المستهدف العديد من التطبيقات في مجال البحوث، بما في ذلك تحليل لتحديد النمط الجينيأدوار الجينات في علم وظائف الأعضاء، وتطوير والمرض. بسبب التسامح المانع للبلمرة DNA المتخصصة، يمكن الانتهاء من البروتوكولات في أقصر وقت ممكن دون تنقية DNA السابقة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> Phire النبات مباشرة PCR كيت </td><td> الحرارية فيشر العلمية </td><td> F-130 </td><td> 200 ردود الفعل (50 ميكرولتر لكل منهما) </td></tr><tr><td> Phire الحيوانية الأنسجة مباشرة PCR كيت </td><td> الحرارية فيشر العلمية </td><td> F-140 </td><td> 200 reations (50 ميكرولتر لكل منهما) </td></tr><tr><td> هاريس أحادي الأساسية 0.35 مم (الوردي) </td><td> الحرارية فيشر العلمية </td><td> F-180S / L </td><td> الكمية: 5/25 </td></tr><tr><td> هاريس أحادي الأساسية 0.50 مم (الأزرق) </td><td> الحرارية فيشر العلمية </td><td> F-185S / L </td><td> الكمية: 5/25 </td></tr><tr><td> هاريس قطع بساط 6.4 × 7.6 سم </td><td> الحرارية فيشر العلمية </td><td> F-190 </td><td> الكمية: 5 </td></tr><tr><td> SSPI </td><td> الحرارية فيشر العلمية </td><td> ER0771 </td><td> 100 ميكرولتر (100 ردود الفعل) </td></tr><tr><td> Piko 24-حسنا الحرارية Cycler </td><td> الحرارية فيشر العلمية </td><td> TCP0024 </td><td></td></tr><tr><td> 24-PCR لوحات حسنا Piko </td><td> الحرارية فيشر العلمية </td><td> SLP0241 </td><td></td></tr><tr><td> GeneJET PCR تنقية كيت </td><td> الحرارية فيشر العلمية </td><td> K0701 K0702 </td><td> 50 محضرات 250 محضرات </td></tr></tbody></table>

genotyping of plant and animal samples without prior dna purification

النهج PCR التضخيم PCR المباشرة يسهل مباشرة من كميات صغيرة من عينات غير منقى، ويتجلى هنا لمحطة عدة والأنسجة الحيوانية (الشكل 1). ويستند مباشرة PCR على تصميم هندسي خاص Phusion العلمية الحرارية وبلمرة DNA Phire، والتي تشمل مجال الحمض النووي المزدوج تقطعت بهم السبل الملزمة التي يعطيها خصائص فريدة مثل التسامح عالية من مثبطات. PCR المستندة إلى الكشف عن الحمض النووي لديها العديد من التطبيقات المستهدفة في مجال البحوث النباتية، بما في ذلك تحليل الوراثي النباتي والتحقق من جينات منقولة. PCR من الأنسجة النباتية ينطوي عادة على عزل الحمض النووي الأولية الخطوة، والتي قد تتطلب الكواشف باهظة الثمن أو السامة. هذه العملية تستغرق وقتا طويلا ويزيد من مخاطر التلوث عبر 1، 2. وعلى العكس، يمكن باستخدام الحرارية العلمية Phire النبات مباشرة PCR كيت يكون الهدف الكشف عن DNA بسهولة، دون استخراج الحمض النووي قبل. في نموذج أثبت هنا، وهويتم تنفيذ مثال المستمدة المشقوق تضخيم تحليل تسلسل متعدد الأشكال (dCAPS) 3،4 مباشرة من أوراق نبتة Arabidopsis. ويمكن استخدام المقايسات dCAPS التنميط الجيني لتحديد الأشكال المتعددة للتركيبات النووية المنفردة (النيوكلوتايد) من أليل محددة SNP تقييد نوكلياز داخلية الهضم 3. بعض العينات النباتية تميل إلى أن تكون أكثر تحديا عند استخدام أساليب المباشر PCR لأنها تحتوي على المكونات التي تتداخل مع PCR، مثل المركبات الفينولية. في هذه الحالات، يلزم عادة خطوة إضافية لإزالة مركبات 2،5. هنا، يتم التغلب على هذه المشكلة باستخدام بروتوكول التخفيف السريع والسهل PCR التضخيم تليها مباشرة (الشكل 1). وتستخدم خمسة عشر أوراق البلوط عاما نموذجا للتحدي والنباتات العينة تحتوي على كميات عالية من المركبات الفينولية بما في ذلك العفص. ويستخدم على نطاق واسع نقل الجينات في الفئران لدراسة دور الجينات في development، علم وظائف الأعضاء والأمراض التي تصيب البشر. استخدام هذه الحيوانات يتطلب الكشف عن وجود التحوير، وعادة مع PCR. تقليديا، وهذا ينطوي على الوقت العزلة طويلا DNA خطوة خلالها DNA لتحليل PCR يتم تنقيته من أنسجة الأذن والذيل أو أخمص القدمين 6،7. ومع ذلك، يمكن مع العلم الحرارية الأنسجة الحيوانية Phire المباشر الفئران المعدلة وراثيا PCR كيت أن التنميط الجيني DNA دون تنقية السابقة. في هذا التنميط الجيني بروتوكول الماوس المعدلة وراثيا ويتحقق مباشرة من أنسجة الأذن الماوس، كما يتضح هنا للحصول على سبيل المثال صعبة حيث يتم استخدام مجموعة واحدة فقط التمهيدي لتضخيم اثنين من شظايا مختلفة اختلافا كبيرا في الحجم.

Watch this Scientific Journal Video about Genotyping of Plant and Animal Samples without Prior DNA Purification at JoVE.com

التنميط الجيني للعينات النباتية والحيوانية دون تنقية الحمض النووي مسبقًا | الأحياء | فيديو

Genotyping of Plant and Animal Samples without Prior DNA Purification

النهج PCR المباشرة المقدمة هنا يسهل PCR التضخيم مباشرة من المصنع كميات صغيرة من أنسجة الحيوانات وغير منقى.

التنميط الجيني للعينات النباتية والحيوانية دون تنقية DNA قبل

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

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30.9K Views.  Thermo Fisher Scientific. النهج PCR المباشرة المقدمة هنا يسهل PCR التضخيم مباشرة من المصنع كميات صغيرة من أنسجة الحيوانات وغير منقى.

Video: Genotyping of Plant and Animal Samples without Prior DNA Purification

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

تلطيخ من البروتينات في الهلام مع Coomassie G - 250 دون عضوي حمض الخليك والمذيبات

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

تبسيط تنقية DNA البلازميد من ثقافات بروكاريوتيك

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

والطريقة انماط PCR القائم على التمييز بين الأنواع البرية من نوع والزينة لل Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

تنقية انجاس: تسلسل Metagenomes وMetatranscriptomes من مجمع عينات المرتبط الحيوانية

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

التضخيم، الجيل التالي من التسلسل، والحمض النووي الجينوم رسم خرائط مواقع التكامل فيروسات الرجعية

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

الإنتاجية العالية كريسبر ناقل البناء وتوصيف التعديلات الحمض النووي عن طريق توليد جذور الطماطم مشعر

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

تنقية الجينوم على نطاق خارج الصبغي التعميم الحمض النووي من خلايا حقيقية النواة

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

الهجين قطع: طريقة محسنة باجتزاء لعينات الأنسجة النباتية المتمردة

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

مسح بروتوكولات المجهر الإلكتروني الماسح (SEM) لإشكالية النبات، طلائعيات بيضية، وعينات الفطرية

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

عزل الحمض النووي الريبي الخاص بنوع الخلية من عينات الأنسجة غير المتجانسة المجمدة بدون فرز الخلايا

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...