לדגימות ארבידופסיס וprimers PCR המודאג אנו מודים פרוף Jaakko Kangasjärvi וקבוצתו, קבוצת לחץ הצמח, ביולוגית צמח, המחלקה לBiosciences, אוניברסיטת הלסינקי. הניסויים עם השיטה המסורתית שנערכו על ידי הגב &#39;Airi Lamminmäki. דגימות עכבר מהונדסות נמסרו על ידי ד&quot;ר יאנה Vesterinen, מכון לביו / ביוכימיה, אוניברסיטת הלסינקי. אריס Uni-Core ומאט חיתוך האריס הם סימנים מסחריים של Shunderson התקשורת בע&quot;מ כל הסימנים המסחריים האחרים הם הרכוש של תרם הפישר סיינטיפיק בע&quot;מ וחברות הבנות שלה.

<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>

הייצור והגישה חופשיה למאמר זה הוא בחסות תרם פישר סיינטיפיק, Inc

הגישה הישירה PCR הדגימה כאן מאפשרת להגברת PCR ישירות מכמויות קטנות של דגימות unpurified, צמצום הזמן הנדרש ולפשט את זרימת העבודה למפעל וgenotyping בעלי החיים (איור 7). הוכיח גם כאן הוא השימוש בפרוטוקול הדילול בשילוב עם הגישה הישירה PCR (איור 3). פרוטוקול הדילול מומלץ עם דגימות עלים קשות (למשל בגיל צמח, מיני צמחים המכילים תרכובות מפריעות) או עם amplicons הארוך (או GC-עשיר). פרוטוקול זה שימושי במיוחד כאשר מתחיל ניסוי חדש ישיר PCR, המאפשר אופטימיזציה של התגובה. הפרוטוקול יכול לשמש ככלי לטיפול בקשיים מזדמנים כגון תשואות מוצר נמוכות בשל הבדלים בחומר ברקמות ו / או פריימרים המשומשים. יתר על כן, בעת הרצת תגובות מרובות מאותו המדגם, שימוש בפרוטוקול הדילול מבטיח כל המדגם אינו נצרך באחדתגובה. כדי לטהר את ה-DNA מצמחים לPCR, הכנס היה לבצע תמוגה תא אחרי בידוד DNA תוך שימוש ברכיבים שונים שעלולים להפריע לניתוח PCR 8. לצמחים ובמיוחד מאתגרים עם תוכן פנוליות גבוה, תוספת של polyvinylpyrrolidone שמש באופן מסורתי כדי להסיר את התרכובות שנקשרות ל-DNA בעקבות תמוגה תא 2,5. כפי שניתן לראות כאן, ניתן להימנע מפעולות המסובכות האלה, זמן עתירות באמצעות שימוש בצמח Phire הישירה PCR הערכה לזיהוי DNA היעד. תמוגה תא וצעדי בידוד DNA ניתן להשמיט תוך שימוש בטכניקות dCAPS הרגישות ישירות מעלי צמח ארבידופסיס (איור 2). כפי שהודגם כאן, פרוטוקול דילול יעילות מנהל את הנוכחות של מרכיבים בעייתיים שיכול להפריע PCR (איור 3). באופן מסורתי, תנאי מוקדם לבעלי החיים genotyping היה isolatiעל של הדנ&quot;א הגנומי מרקמות בעלי חיים באמצעות תהליך רעיל, זמן רב המאופיינים בדגירה ארוכה עם proteinase K לעכל עור ורקמת חיבור, ואחרי חילוץ ממס אורגני, משקעי אלכוהול, וצעדי צנטריפוגה 6,9,10. כפי שהודגם כאן, פישוט תהליך זה מושג באמצעות שימוש בבעלי חי Phire רקמות הישירות PCR הקיט, המאפשר עכברים הטרנסגניים להיות genotyped ללא טיהור DNA לפני (איור 4). הדוגמא הראתה במאמר זה בעיקר מאתגרת כמו שרק סט אחד פריימר שמש להגברה של שני שברים עם הבדל בגודל גדול. למרות הקושי המולד של הפרוטוקול, שימוש בגישה הישירה PCR בשיתוף עם פרוטוקול הדילול פשוט הביא תשואות גבוהות של שני מוצרי PCR (איור 4). זיהוי DNA יעד PCR מבוסס יש יישומים רבים במחקר, כולל ניתוח גנוטיפ לזהותאת התפקידים של גנים בהתפתחות, פיזיולוגיה ומחלות. בשל סובלנות המעכב של polymerases דנ&quot;א המיוחד, את הפרוטוקולים ניתן להשלים בזמן מינימאלי ללא טיהור DNA מוקדמת.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> Phire צמח הישיר PCR Kit </td><td> תרם הפישר סיינטיפיק </td><td> F-130 </td><td> 200 תגובות (50 μl כל אחד) </td></tr><tr><td> Phire בעלי חי רקמות הישירה PCR Kit </td><td> תרם הפישר סיינטיפיק </td><td> F-140 </td><td> 200 reations (50 μl כל אחד) </td></tr><tr><td> אריס Uni-Core 0.35 מ&quot;מ (ורוד) </td><td> תרם הפישר סיינטיפיק </td><td> F-180S / L </td><td> כמות: 5/25 </td></tr><tr><td> אריס Uni-Core 0.50 מ&quot;מ (כחול) </td><td> תרם הפישר סיינטיפיק </td><td> F-185S / L </td><td> כמות: 5/25 </td></tr><tr><td> אריס חיתוך Mat 6.4 × 7.6 סנטימטר </td><td> תרם הפישר סיינטיפיק </td><td> F-190 </td><td> כמות: 5 </td></tr><tr><td> SSPI </td><td> תרם הפישר סיינטיפיק </td><td> ER0771 </td><td> 100 μl (100 תגובות) </td></tr><tr><td> Piko Cycler 24 ובכן התרמית </td><td> תרם הפישר סיינטיפיק </td><td> TCP0024 </td><td></td></tr><tr><td> צלחות PCR Piko 24 Well </td><td> תרם הפישר סיינטיפיק </td><td> SLP0241 </td><td></td></tr><tr><td> GeneJET PCR ערכת טיהור </td><td> תרם הפישר סיינטיפיק </td><td> K0701 K0702 </td><td> 50 preps 250 preps </td></tr></tbody></table>

genotyping of plant and animal samples without prior dna purification

הגישה הישירה PCR מאפשרת הגברת PCR ישירות מכמויות קטנות של דגימות unpurified, והוא הוכיח כאן לכמה צמח ורקמות של חיות (איור 1). הישיר PCR מבוסס על הונדס במיוחד Thermo Scientific polymerases DNA Phire Phusion ו, הכולל תחום מחייב גדילי הדנ&quot;א כפול שנותן להם תכונות ייחודיות כגון סובלנות גבוהה של מעכבים. זיהוי DNA יעד PCR מבוסס יש יישומים רבים במחקר במפעל, כולל ניתוח ובדיקה של transgenes גנוטיפ צמח. PCR מרקמות צמח באופן מסורתי כרוך שלב ראשוני DNA בידוד, שעשוי לדרוש ריאגנטים יקרים או רעילים. התהליך דורש זמן ומגביר את הסיכון לזיהום צולב 1, 2. לעומת זאת, על ידי שימוש Thermo Scientific Phire צמח ישיר PCR ערכת DNA היעד ניתן לאתר בקלות, ללא מיצוי DNA מוקדם. במודל הפגין כאן,דוגמה לניתוח שנגזר בקע מוגבר רב צורות רצף (dCAPS) 3,4 מתבצעת ישירות מעלי הצמח ארבידופסיס. מבחני dCAPS genotyping יכולים לשמש לזיהוי פולימורפיזמים נוקלאוטיד בודדים (SNPs) על ידי עיכול SNP אלל ספציפי הגבלת endonuclease 3. כמה דוגמאות צמחים נוטות להיות מאתגר יותר בעת שימוש בשיטות PCR ישירות כפי שהם מכילים רכיבים שמפריעים לPCR, כמו תרכובות פנוליות. במקרים אלה, צעד נוסף כדי להסיר את התרכובות נדרש 2,5 באופן מסורתי. כאן, בעיה זו ניתן להתגבר על ידי שימוש בפרוטוקול דילול קל ומהיר ואחרי הגברת PCR ישירה (איור 1). חמש העשרה אלון בן משמשים כמודל עבור צמחים מאתגרים, כדגם מכיל כמויות גבוהות של תרכובות פנוליות כוללים טאנינים. העברת גנים לעכברים משמשת באופן נרחב כדי ללמוד את התפקידים של גנים בdevelopment, פיזיולוגיה ומחלות של בני אדם. השימוש בבעלי החיים אלה מחייב בדיקות לנוכחות של transgene, בדרך כלל עם ה-PCR. באופן מסורתי, זה כרוך בצעד זמן רב DNA בידוד, שבמהלכו DNA לניתוח PCR מטוהר מרקמות אוזן, זנב או בוהן 6,7. עם זאת, עם עכברי Thermo Scientific Phire בעלי חי הרקמות הישירות PCR Kit המהונדסים ניתן genotyped ללא טיהור DNA מוקדמת. בפרוטוקול המהונדס העכבר genotyping זה הושג ישירות מרקמות אוזן עכבר, כפי שמודגם כאן לדוגמה מאתגרת שבו רק סט אחד פריימר משמש להגברה של שני שברים שונים מאוד בגודלו.

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

גנוטיפ של דגימות צמחים ובעלי חיים ללא טיהור DNA קודם | ביולוגיה | וִידֵאוֹ

Genotyping of Plant and Animal Samples without Prior DNA Purification

הגישה הישירה PCR מוצגת כאן מאפשרת הגברת PCR ישירות מכמויות קטנות של צמח unpurified ורקמות בעלי חיים.

Genotyping של דוגמאות צמחים ובעלי חיים, ללא טיהור DNA לפני

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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31.1K Views.  Thermo Fisher Scientific. הגישה הישירה PCR מוצגת כאן מאפשרת הגברת PCR ישירות מכמויות קטנות של צמח unpurified ורקמות בעלי חיים.

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.

הכתמה של חלבונים עם ג&#39;ל Coomassie G-250 ללא חומצה אצטית ממס אורגני

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.

טיהור יעיל של ה-DNA פלסמיד מתרבויות פרוקריוטים

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).

PCR מבוסס genotyping שיטה להבחין בין זנים wild-type ו נוי של 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.

טיהור הלא טהור: Metagenomes הרצף וMetatranscriptomes מדוגמאות הקשורים בבעלי חיים מורכבות

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.

הגברה, רצף הדור הבא, ואת הגנום מיפוי ה- DNA של אתרי אינטגרצית retroviral

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.

CRISPR וקטור בינוי אפיון תפוקה גבוהה של שינויי DNA על ידי דור של שורשי עגבניות שעירים

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.

טיהור הגנום כולו של DNA המעגל extrachromosomal מתאי איקריוטיים

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).

-Cut ההיברידי: שיטת חתך משופרת עבור דוגמאות רקמות צמח סרבן

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.

מיקרוסקופ אלקטרוני סורק הפרוטוקולים (SEM) עבור בעייתי הצמח, Oomycete, וכן דוגמאות פטרייתיים

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

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Genotyping של דוגמאות צמחים ובעלי חיים, ללא טיהור DNA לפני

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