מחקר זה נתמך בחלקו על ידי המכון הלאומי לבריאות (GM071440 לCH וNS051630/MH076090/MH078972 לPJ).

1. פיצול הדנ&quot;א הגנומי הדנ&quot;א הגנומי בר באמצעות sonication לטווח גודל רצוי מתאים לפלטפורמת רצף הגנום כולו. (בדרך כלל אנחנו sonicate ל ~ 300 נ&quot;ב.) ודא התפלגות הגודל של הדנ&quot;א הגנומי המקוטע ב% agarose ג&#39;ל 1 (1 איור). 2. הכנת דנ&quot;א לקבוע את הסכומים המבוססים על דנ&quot;א החל השפע של 5-HMC בהדנ&quot;א הגנומי. מאז רמות 5-HMC להשתנות באופן משמעותי בסוגי רקמות שונים, כמויות DNA החל תלויות ברמות 5-HMC של הדגימות. אנא עיין בטבלת המס &#39;1 לדוגמאות. 3. תגובה מזורז β-GT (תגובת העברת גלוקוז) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מערבבים ידי pipetting את התערובת כמפורטת בטבלת 2 ולדגור ב° C אמבט מים במשך 37 שעות 1. </li><li style=";text-align:right;direction:rtl"> לאחר דגירה, לנקות תגובה עם ערכה להסרת נוקלאוטיד QIAquick, באמצעות 10 מיקרוגרם של ה-DNA לעמודה. Elute עם מי μl 30 לעמודה ולשלב. </li></ol> 4. תגובת Biotinylation (לחץ כימיה) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף פתרון עובד הצמוד DBCO-SS-PEG3-ביוטין (מ&quot;מ 1) בפתרון ה-DNA eluted (משלב 3) לריכוז סופי של 150 מיקרומטר (כלומר, 5 μl של עובד פתרון לפתרון דנ&quot;א 30 μl). </li><li style=";text-align:right;direction:rtl"> מערבבים ידי pipetting ולדגור ב° C אמבט מי 37 עבור 2 שעות. </li><li style=";text-align:right;direction:rtl"> לנקות תגובה עם ערכה להסרת נוקלאוטיד QIAquick. הנפח הכולל elution האידיאלי הוא 100 μl. </li><li style=";text-align:right;direction:rtl"> לכמת את כמות הדנ&quot;א שנמצא באמצעות ספקטרופוטומטר ההיקף microliter (למשל., NanoDrop). </li></ol> 5. לכידה של 5-DNA המכיל HMC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטוף 50 μl של Dynabeads MyOne streptavidin C1 3 פעמים עם 1 מ&quot;ל של 1X B &amp; W חיץ פי הוראות היצרן. הפרד את החרוזים עם מעמד מגנטי. </li><li style=";text-align:right;direction:rtl"> הוסף נפח שווה של 2X B &amp; W לחיץ התאושש D biotinylatedNA (100 ul) לחרוזים והבהירים. </li><li style=";text-align:right;direction:rtl"> דגירה במשך 15 דקות בטמפרטורת חדר עם סיבוב קל על כתף. </li><li style=";text-align:right;direction:rtl"> הפרד את החרוזים עם מעמד מגנטי ולשטוף את החרוזים 3 פעמים עם 1 מ&quot;ל של 1X B &amp; W חיץ. </li><li style=";text-align:right;direction:rtl"> Elute דנ&quot;א על ​​ידי דוגר את החרוזים ב100 μl של DTT mM המוכן טרי 50 עבור 2 שעות בטמפרטורת חדר עם סיבוב קל על כתף. </li><li style=";text-align:right;direction:rtl"> הפרד את החרוזים עם מעמד מגנטי. לשאוב eluent ועומס על 6 טור מייקר יו ספין בהתאם להוראות הייצור להסיר DTT. DNA היעד הוא בפתרון עכשיו. </li><li style=";text-align:right;direction:rtl"> לטהר את הדנ&quot;א eluted מהשלב הקודם על ידי ערכת Qiagen MinElute PCR DNA וטיהור elute ב10 μl של חיץ EB. לכמת דנ&quot;א באמצעות Fluorometer קיוביט, או NanoDrop אם הריכוז גבוה מ 20 ng / ul. ה-DNA היא מוכנה להכנת ספריית מורד הגנום רצף. </li></ol> 6. נציג תוצאות אם איכות oהדנ&quot;א הגנומי f הוא גבוה, תשואות התאוששות טיפוסיות לאחר β-GT ותגובות biotinylation הן ~ 60-70%. עם זאת, יעילות הלכידה להשתנות באופן משמעותי עם סוגי רקמות שונים בהתאם לרמות 5-HMC של הדגימות. בדרך כלל, את היעילות הלכידה להדנ&quot;א הגנומי מוח היא ~ 4-9%, ובכמה מקרים קיצוניים, היעילות עשויה להגיע עד 12%. לתאי גזע עוברי, יעילות הלכידה הממוצעת היא ~ 2-4%, בניגוד ל~ 0.5% לתאי גזע עצביים. היעילות הנמוכה ביותר שראתה עד כה הייתה להדנ&quot;א הגנומי מתאים סרטניים. כל הדנ&quot;א המועשר מוכן להכנת פרוטוקולים סטנדרטיים של הדור הבא של ספרייה. בנוסף, ה-DNA שנתפס יכול לשמש גם כתבנית לPCR בזמן אמת כדי לזהות את ההעשרה של שברים מסוימים בהשוואה ל-DNA הקלט, אם את פריימרים הקשורים אינם זמינים. <img alt="איור 1" src="/files/ftp_upload/4441/4441fig1.jpg" /> איור 1. ברי sonicated אדם הגנומי DNA ב1% agarose ג&#39;ל. 10 מיקרוגרם של הדנ&quot;א הגנומי המבודד מתאי iPS אדם ב120 μl של 1X TE חיץ היה sonicated באמצעות מכשיר sonication (Covaris). לאחר sonication, 2 μl של דנ&quot;א sonicated הועמס 1% agarose ג&#39;ל באמצעות 100 נ&quot;ב של סמן ה-DNA כדי להשוות את סדר הגודל של מקטעי דנ&quot;א sonicated. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> רכיב </td><td> נפח </td><td> ריכוז סופי </td></tr><tr><td> מים </td><td> _ Μl </td><td></td></tr><tr><td> 10 X הצפת תגובת β-GT </td><td> 2 μl </td><td> 1 X </td></tr><tr><td> עד הדנ&quot;א הגנומי מיקרוגרם 10 </td><td> _ Μl </td><td> עד 500 ng / μl </td></tr><tr><td> UDP-6-3-N GLC (3 מ&quot;מ) </td><td> 0.67 μl </td><td> 100 מיקרומטר </td></tr><tr><td> β-GT (40 מיקרומטר) </td><td> μl 1 </td><td> 2 מיקרומטר </td></tr><tr><td> נפח כולל </td><td> 20 μl </td&gt; <td></td></tr></tbody></table> i) להדנ&quot;א הגנומי רקמות (תכולה גבוהה 5-HMC&gt; 0.1%) <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> רכיב </td><td> נפח </td><td> ריכוז סופי </td></tr><tr><td> מים </td><td> _ Μl </td><td></td></tr><tr><td> 10 X הצפת תגובת β-GT </td><td> 10 μl </td><td> 1 X </td></tr><tr><td> עד הדנ&quot;א הגנומי מיקרוגרם 20 </td><td> _ Μl </td><td> עד 500 ng / μl </td></tr><tr><td> UDP-6-N3-GLC (3 מ&quot;מ) </td><td> μl 1.33 </td><td> 100 מיקרומטר </td></tr><tr><td> β-GT (40 מיקרומטר) </td><td> 2 μl </td><td> 2 מיקרומטר </td></tr><tr><td> נפח כולל </td><td> 40 μl </td><td></td></tr></tbody></table> ii) להדנ&quot;א הגנומי תאי גזע (חציון 5-HMC תוכן ~ 0.05%) <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> רכיב </td><td> נפח </td><td> ריכוז סופי </td></tr><tr><td> מים </td><td> _ Μl </td><td></td></tr><tr><td> 10 X הצפת תגובת β-GT </td><td> 10 μl </td><td> 1 X </td></tr><tr><td> עד הדנ&quot;א הגנומי מיקרוגרם 50 </td><td> _ Μl </td><td> עד 500 ng / μl </td></tr><tr><td> UDP-6-N3-GLC (3 מ&quot;מ) </td><td> 3.33 μl </td><td> 100 מיקרומטר </td></tr><tr><td> β-GT (40 מיקרומטר) </td><td> 5 μl </td><td> 2 מיקרומטר </td></tr><tr><td> נפח כולל </td><td> 100 μl </td><td></td></tr></tbody></table> ג) לדנ&quot;א סרטן התאים גנומית (תוכן נמוך 5-HMC ~ 0.01%) טבלת 1. דוגמאות לסכומים של ה-DNA קלט ותגובות תיוג באמצעות דגימות עם רמות 5-HMC שונים על ידי שיטת התיוג הכימי סלקטיבית. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> מדגם </td><td> רמת 5-HMC </strong&gt; </td><td> דנ&quot;א החל (מיקרוגרם) </td><td> התאוששות לאחר תיוג (קלט לחרוזים) (מיקרוגרם) </td><td> תשואת שחזור </td><td> נפתח דנ&quot;א (נ&quot;ג) </td><td> משוך כלפי מטה תניב </td></tr><tr><td> מוח קטן עכבר בוגר </td><td> 0.4% </td><td> 10 </td><td> 7.5 </td><td> 75% </td><td> 236 </td><td> 3.1% </td></tr><tr><td> מוח קטן עכבר אחרי לידת יום 7 </td><td> 0.1% </td><td> 11 </td><td> 9 </td><td> 82% </td><td> 140 </td><td> 1.6% </td></tr><tr><td> תא עכבר ES E14 </td><td> 0.05% </td><td> 60 </td><td> 42 </td><td> 70% </td><td> 350 </td><td> 0.8% </td></tr></tbody></table> טבלה 2. נציג תוצאות מרקמות מוח עכבר ותאי גזע עוברי.

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אין ניגודי האינטרסים הכריזו.

5-hydroxymethylcytosine (5-HMC) הוא הווה שינוי epigenetic זיהה לאחרונה בכמויות ניכרות בסוגי תאי יונקים מסוימים. השיטה שהוצגה כאן היא לקביעת חלוקת הגנום של 5-HMC. אנו משתמשים bacteriophage T4 β-glucosyltransferase להעביר מחצית גלוקוז מהונדסת המכילה קבוצה תזיד על קבוצת הידרוקסיל של 5-HMC. הקבוצה תזיד יכולה לה...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם </td><td> חברה </td><td> קטלוג # </td><td> הערה </td></tr><tr><td></td><td></td><td></td><td> ריאגנטים </td></tr><tr><td> נתרן כלורי 5M (NaCl) </td><td> Promega </td><td> V4221 </td><td></td></tr><tr><td> חומצת ethylenediaminetetraacetic pH8.0 0.5m (EDTA) </td><td> Promega </td><td> V4231 </td><td></td></tr><tr><td> 1M Trizma בסיס (טריס) pH7.5 </td><td> Invitrogen </td><td> 15567-027) </td><td></td></tr><tr><td> HEPES 1M, pH7.4 </td><td> Invitrogen </td><td> 15630 </td><td></td></tr><tr><td> מגנזיום כלוריד (MgCl 2) 1M </td><td> Ambion </td><td> AM9530 </td><td></td></tr><tr><td> sulfoxide דימתיל (DMSO) </td><td> סיגמא </td><td> D8418 </td><td></td></tr><tr><td> 20 Tween </td><td> BioReagents הפישר </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3-ביוטין הצמוד </td><td> לחץ על כלי כימיה </td><td> A112P3 </td><td></td></tr><tr><td> 1,4-Dithiothreitol, ultrapure (DTT) Superpure </td><td> Invitrogen </td><td> 15508-013 </td><td></td></tr><tr><td> ערכה להסרת נוקלאוטיד QIAquick </td><td> Qiagen </td><td> 28304 </td><td></td></tr><tr><td> מייקרו יו ספין 6 טור </td><td> Bio-Rad </td><td> 732-6222 </td><td></td></tr><tr><td> Dynabeads MyOne </td><td> Invitrogen </td><td> 650-01 </td><td></td></tr><tr><td> Streptavidin C1 </td><td></td><td></td><td></td></tr><tr><td> ערכת טיהור PCR MinElute Qiagen </td><td> Qiagen </td><td> 28004 </td><td></td></tr><tr><td> Ultrapure agarose </td><td> Invitrogen </td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-3-N גלוקוז </td><td> מוטיב פעיל </td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> אנזים </td></tr><tr><td> β-glucosyltransferase (β-GT) </td><td> ניו אינגלנד Biolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> ציוד </td></tr><tr><td> מכשיר sonication </td><td> Covaris </td><td></td><td></td></tr><tr><td> צנטריפוגה שולחן העבודה </td><td></td><td></td><td></td></tr><tr><td> אמבט מים </td><td> הפישר סיינטיפיק </td><td></td><td></td></tr><tr><td> מנגנון פועל ג&#39;ל </td><td> Bio-Rad </td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Labquake Tube אכר </td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquake Tube אכר </td><td> Thermolyne </td><td></td><td></td></tr><tr><td> סטנד הפרדה מגנטי </td><td> Promega </td><td> Z5342 </td><td></td></tr><tr><td> קיוביט 2.0 Fluorometer </td><td> Invitrogen </td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> התקנת מגיב 10 X תגובת β-GT מאגר (500 HEPES המ&quot;מ pH 7.9, 250 מ&quot;מ MgCl 2) 2 X עקידה וכביסת חיץ (B &amp; W) (10 mM טריס pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% 20 Tween) . </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5-methylcytosine (5-MC) מהווה ~ 2-8% מכלל cytosines בהדנ&quot;א הגנומי אדם ומשפיע על מגוון רחב של תפקודים ביולוגיים, כולל ביטוי גנים, שמירה על שלמות הגנום, החתמה הורית, איון כרומוזום ה-X, רגולציה של פיתוח, הזדקנות וסרטן 1. לאחרונה, הנוכחות של חומץ 5-MC, 5-hydroxymethylcytosine (5-HMC), התגלתה בתאי יונקים, ובעיקר בתאי גזע עובריים (ES) ותאים עצביים 2-4. 5-HMC נוצר על ידי חמצון של 5-MC קטליזאטור ידי ט משפחת הברזל (ב &#39;) / dioxygenases α-ketoglutarate התלוי 2, 3. 5-HMC מוצע להיות מעורב בתחזוקה של תא גזע עוברי (MES), hematopoiesis וממאירויות הרגילים, והזיגוטה פיתוח 2, 5-10. כדי להבין טוב יותר את התפקוד של 5-HMC, מערכת רצף אמינה וישירה היא חיונית. רצף יסולפיט מסורתי לא מבחין בין 5-5-HMC מMC 11 </sup&gt;. כדי להסביר את הביולוגיה של 5-HMC, פתח גישה כימית יעילה מאוד סלקטיבית לתייג וללכוד 5-HMC, תוך ניצול של אנזים bacteriophage שמוסיף מחצית סוכר ל5-HMC במיוחד 12. כאן אנו מתארים הליך פשוט שני שלבים לתיוג כימי סלקטיבית של 5-HMC. בשלב התיוג 1, 5-HMC בהדנ&quot;א הגנומי מתויג עם קטליזאטור 6-יזיד-גלוקוז על ידי β-GT, glucosyltransferase מbacteriophage T4, באופן שמעביר את 6-תזיד-גלוקוז ל5-HMC מ העדכון cofactor, UDP-6-N3-GLC (6-N3UDPG). בצעד, biotinylation השני, מקשר ביוטין דיסולפיד מצורף לקבוצה יזיד ידי לחץ כימיה. שני פעולות ספציפיות ויעילות ביותר, שמובילות לתיוג להשלים ללא קשר לשפע של 5-HMC באזורים גנטיים ונותנים רקע נמוך ביותר. בעקבות biotinylation של 5-HMC, שברי 5-DNA המכיל HMC אז הם כבשו סלקטיביבאמצעות חרוזי streptavidin באופן עצמאי בצפיפות. שברי 5-HMC מועשרי DNA המתקבלים יכולים לשמש עבור ניתוחים במורד זרם, כוללים רצף של הדור הבא. התיוג סלקטיבית שלנו ופרוטוקול לכידה מקנים רגישות גבוהה, החלה על כל מקור של הדנ&quot;א הגנומי עם שכיחות משתנית / 5-HMC מגוון. אמנם המטרה העיקרית של פרוטוקול זה היא יישומה במורד הזרם (כלומר., הדור הבא של רצף למפות את התפלגות 5-HMC בגנום), זה תואם עם מולקולה בודדה, SMRT (DNA) רצף בזמן אמת, שהוא מסוגל לספק רזולוצית רצף יחיד בסיס של 5-HMC.

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

תאר הוא תהליך תיוג שני שלבי שימוש (β-GT) β-glucosyltransferase להעביר תזיד-גלוקוז ל5-HMC, ואחרי לחץ כימיה להעביר מקשר ביוטין להעשרה קלה וצפיפות בלתי תלויה. שיטת תיוג יעילה וספציפית זו מאפשרת העשרה של 5-HMC עם רקע נמוך ביותר ומיפוי epigenomic תפוקה גבוהה באמצעות רצף של הדור הבא.

לכידה סלקטיבית של 5-hydroxymethylcytosine מהדנ&quot;א הגנומי

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

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Research

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Biology

11.9K Views.  Emory University School of Medicine. תאר הוא תהליך תיוג שני שלבי שימוש (β-GT) β-glucosyltransferase להעביר תזיד-גלוקוז ל5-HMC, ואחרי לחץ כימיה להעביר מקשר ביוטין להעשרה קלה וצפיפות בלתי תלויה. שיטת תיוג יעילה וספציפית זו מאפשרת העשרה של 5-HMC עם רקע נמוך ביותר ומיפוי epigenomic תפוקה גבוהה באמצעות רצף של הדור הבא.

Video: Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Isolation of Genomic DNA from Mouse Tails

בידוד של הדנ&quot;א הגנומי של זנבות עכבר

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

הכנס גדול הסביבה הגנום ספריית הפקה

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

הפקת DNA גבוהה מולקולרית משקל הגנום מקרקעות ו משקעים

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

הארכת לכידת פריימר: אחזור רצף ממוקד ממקורות DNA מושפל בכבדות

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

הדור של RNA / DNA כלאיים ב-DNA הגנום על ידי טרנספורמציה באמצעות RNA המכיל oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

הגנום MRI - משאב ציבורי עבור לימוד דפוסי בתוך רצף הדנ&quot;א הגנומי

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

מסכי הגנום תחרותי של ספריות שמרים המבורקדים

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

זיהוי גנטי של וריאנטים נדירים מן הרצף נקווה שימוש Splinter

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

שיטה מעשית ורומן לחלץ הדנ&quot;א הגנומי מערכות דם אוסף לשימור חלבון פלזמה

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

הדמיה בזמן אמת של יחיד תכנון הנדסי RNA תמלילים בתאים חיים באמצעות משואות bimolecular רציומטרי

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...