עבודה זו נתמכה על ידי NIH מענקי GM45916 וGM66834 לJCH ומלגה מהקרן הבינלאומית תסמונת רט לAK עבודה זו נתמכה גם על ידי מענק NIH GM088409 והמכון רפואי הווארד יוז תרומות לKL

<ol>
	<li>Luger, K., Mader, A., Richmond, R., Crystal Sargent, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 7, (1997).</li><li>Hansen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+dynamics+of+the+chromatin+fiber+in+solution:+determinants,+mechanisms,+and+functions.">Conformational dynamics of the chromatin fiber in solution: determinants, mechanisms, and functions.</a> Annual Review of Biophysics and Biomolecular Structure. 31, 361-392 (2002).</li><li>McBryant, S., Adams, V., Hansen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+architectural+proteins.">Chromatin architectural proteins.</a> Chromosome Research. 14 (1), 39-51 (2006).</li><li>Hansen, J. C., Ausio, J., Stanik, V. H., van Holde, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+reconstituted+oligonucleosomes,+evidence+for+salt-dependent+folding+in+the+absence+of+histone+H1.">Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1.</a> Biochemistry. 28 (23), 9129-9136 (1989).</li><li>Szerlong, H. J., Prenni, J. E., Nyborg, J. K., Hansen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activator-dependent+p300+acetylation+of+chromatin+in+vitro:+enhancement+of+transcription+by+disruption+of+repressive+nucleosome-nucleosome+interactions.">Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome-nucleosome interactions.</a> The Journal of Biological Chemistry. 285 (42), 31954-31964 (2010).</li><li>Cirillo, L. A., Lin, F. R., Cuesta, I., Friedman, D., Jarnik, M., Zaret, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opening+of+compacted+chromatin+by+early+developmental+transcription+factors+HNF3+(FoxA)+and+GATA-4.">Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.</a> Molecular Cell. 9 (2), 279-289 (2002).</li><li>Nguyen, C., Gonzales, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=chromatin+structure+associated+with+methylation-induced+gene+silencing+in+cancer+cells:+correlation+of+accessibility,+methylation,+MeCP2+binding+and+acetylation.">chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation.</a> Nucleic Acids Research. 29 (22), 4598-4606 (2001).</li><li>Cuesta, I., Zaret, K. S., Santisteban, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forkhead+factor+FoxE1+binds+to+the+thyroperoxidase+promoter+during+thyroid+cell+differentiation+and+modifies+compacted+chromatin+structure.">The forkhead factor FoxE1 binds to the thyroperoxidase promoter during thyroid cell differentiation and modifies compacted chromatin structure.</a> Molecular and Cellular Biology. 27 (20), 7302-7314 (2007).</li><li>Simpson, R. T., Stafford, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+features+of+a+phased+nucleosome+core+particle.">Structural features of a phased nucleosome core particle.</a> Proceedings of the National Academy of Sciences of the U S A. 80 (1), 51-55 (1983).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> Journal of Molecular Biology. 276 (1), 19-42 (1998).</li><li>Lowary, P., Widlund, H., Cao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+motifs+and+free+energies+of+selected+natural+and+non-natural+nucleosome+positioning+DNA+sequences.">Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences.</a> Journal of Molecular Biology. 288, 213-229 (1999).</li><li>Gordon, F., Luger, K., Hansen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Core+Histone+N-terminal+Tail+Domains+Function+Independently+and+Additively+during+Salt-dependent+Oligomerization+of+Nucleosomal+Arrays+*.">The Core Histone N-terminal Tail Domains Function Independently and Additively during Salt-dependent Oligomerization of Nucleosomal Arrays *.</a> The Journal of Biological Chemistry. 280 (40), 33701-33706 (2005).</li><li>Luger, K., Rechsteiner, T. J., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+recombinant+histones+and+nucleosome+reconstitution.">Expression and purification of recombinant histones and nucleosome reconstitution.</a> Methods in Molecular Biology (Clifton, N.J.). 119 (4), 1-16 (1999).</li><li>McBryant, S. J., Klonoski, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+histone+H4+N-terminal+domain+function+during+nucleosomal+array+oligomerization:+roles+of+amino+acid+sequence,+domain+length,+and+charge+density.">Determinants of histone H4 N-terminal domain function during nucleosomal array oligomerization: roles of amino acid sequence, domain length, and charge density.</a> The Journal of Biological Chemistry. 284 (25), 16716-16722 (2009).</li><li>Ma Shogren-Knaak, ., Fry, C. J., Peterson, C. 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+native+peptide+ligation+strategy+for+deciphering+nucleosomal+histone+modifications.">A native peptide ligation strategy for deciphering nucleosomal histone modifications.</a> The Journal of Biological Chemistry. 278 (18), 15744-158 (2003).</li><li>Lu, X., Simon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+H3K79+dimethylation+and+H4K20+trimethylation+on+nucleosome+and+chromatin+structure.">The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structure.</a> Nat Struct Mol Biol. 15 (10), 1122-1124 (2008).</li><li>Ausio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+Ultracentrifugation+for+the+Analysis+of+Chromatin+Structure.">Analytical Ultracentrifugation for the Analysis of Chromatin Structure.</a> Biophysical Chemistry. 86 (2-3), 141-153 (2000).</li><li>Hansen, J., Kreider, J., Demeler, B., Fletcher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+ultracentrifugation+and+agarose+gel+electrophoresis+as+tools+for+studying+chromatin+folding+in+solution.">Analytical ultracentrifugation and agarose gel electrophoresis as tools for studying chromatin folding in solution.</a> Methods. 12 (1), 62-72 (1997).</li><li>Montel, F., Menoni, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+individual+nucleosomes+controls+the+chromatin+condensation+pathway:+direct+atomic+force+microscopy+visualization+of+variant+chromatin.">The dynamics of individual nucleosomes controls the chromatin condensation pathway: direct atomic force microscopy visualization of variant chromatin.</a> Biophysical Journal. 97 (2), 544-5453 (2009).</li><li>Muthurajan, U. M., McBryant, S. J., Lu, X., Hansen, J. C., Luger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linker+region+of+macroH2A+promotes+self-association+of+nucleosomal+arrays.">The linker region of macroH2A promotes self-association of nucleosomal arrays.</a> The Journal of Biological Chemistry. 286 (27), 23852-23864 (2011).</li><li>Shlyakhtenko, L. S., Lushnikov, A. Y., Lyubchenko, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+nucleosomes+revealed+by+time-lapse+atomic+force+microscopy.">Dynamics of nucleosomes revealed by time-lapse atomic force microscopy.</a> Biochemistry. 48 (33), 7842-7848 (2009).</li><li>Lohr, D., Bash, R., Wang, H., Yodh, J., Lindsay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+atomic+force+microscopy+to+study+chromatin+structure+and+nucleosome+remodeling.">Using atomic force microscopy to study chromatin structure and nucleosome remodeling.</a> Methods (San Diego, Calif). 41 (3), 333-341 (2007).</li><li>Dyer, P. N., Edayathumangalam, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+nucleosome+core+particles+from+recombinant+histones+and+DNA.">Reconstitution of nucleosome core particles from recombinant histones and DNA.</a> Methods in Enzymology. 375, 23-44 (2004).</li><li>Sambrook, J., Russell, D. <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 manual. , (2001).</li><li>Balbo, A., Zhao, H., Brown, P. H., Schuck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly,+loading,+and+alignment+of+an+analytical+ultracentrifuge+sample+cell.">Assembly, loading, and alignment of an analytical ultracentrifuge sample cell.</a> J. Vis. Exp. (33), e1530 (2009).</li><li>Demeler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UltraScan:+a+comprehensive+data+analysis+software+package+for+analytical+ultracentrifugation+experiments.">UltraScan: a comprehensive data analysis software package for analytical ultracentrifugation experiments.</a> Modern Analytical Ultracentrifugation: Techniques. , 210-230 (2005).</li><li>Holde, K. V., Weischet, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boundary+analysis+of+sedimentation+velocity+experiments+with+monodisperse+and+paucidisperse+solutes.">Boundary analysis of sedimentation velocity experiments with monodisperse and paucidisperse solutes.</a> Biopolymers. 17 (6), 1387-1403 (1978).</li><li>Demeler, B., van Holde, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sedimentation+velocity+analysis+of+highly+heterogeneous+systems.">Sedimentation velocity analysis of highly heterogeneous systems.</a> Analytical Biochemistry. 335, 279-288 (2004).</li><li>Hansen, J., Lohr, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+structural+properties+of+subsaturated+chromatin+arrays.">Assembly and structural properties of subsaturated chromatin arrays.</a> Journal of Biological Chemistry. 8, 5840-5848 (1993).</li><li>Routh, A., Sandin, S., Rhodes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+repeat+length+and+linker+histone+stoichiometry+determine+chromatin+fiber+structure.">Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure.</a> Proceedings of the National Academy of Sciences of the U S A. 105 (26), 8872-8877 (2008).</li><li>Zhou, J., Fan, J. Y., Rangasamy, D., Tremethick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleosome+surface+regulates+chromatin+compaction+and+couples+it+with+transcriptional+repression.">The nucleosome surface regulates chromatin compaction and couples it with transcriptional repression.</a> Nature Structural &amp; Molecular Biology. 14 (11), 1070-1076 (2007).</li><li>Dorigo, B., Schalch, T., Kulangara, A., Duda, S., Schroeder, R. R., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+arrays+reveal+the+two-start+organization+of+the+chromatin+fiber.">Nucleosome arrays reveal the two-start organization of the chromatin fiber.</a> Science (New York, N.Y.). 306 (5701), 1571-1573 (2004).</li><li>Correll, S. J., Schubert, M. H., Grigoryev, 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+Short+nucleosome+repeats+impose+rotational+modulations+on+chromatin+fibre+folding.">a Short nucleosome repeats impose rotational modulations on chromatin fibre folding.</a> The EMBO Journal. 31 (10), 2416-2426 (2012).</li><li>Mcbryant, S. J., Krause, C., Woodcock, C. L., Hansen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Silent+Information+Regulator+3+Protein+,+SIR3p+,+Binds+to+Chromatin+Fibers+and+Assembles+a+Hypercondensed+Chromatin+Architecture+in+the+Presence+of+Salt.">The Silent Information Regulator 3 Protein , SIR3p , Binds to Chromatin Fibers and Assembles a Hypercondensed Chromatin Architecture in the Presence of Salt.</a> Molecular and Cellular Biology. 28 (11), 3563-3572 (2008).</li><li>Schalch, T., Duda, S., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+structure+of+a+tetranucleosome+and+its+implications+for+the+chromatin+fibre.">X-ray structure of a tetranucleosome and its implications for the chromatin fibre.</a> Nature. 436 (7047), 138-1341 (2005).</li><li>Fan, J. Y., Gordon, F., Luger, K., Hansen, J. C., Tremethick, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+histone+variant+H2A.Z+regulates+the+equilibrium+between+different+chromatin+conformational+states.">The essential histone variant H2A.Z regulates the equilibrium between different chromatin conformational states.</a> Nature Structural Biology. 9 (3), 172-176 (2002).</li><li>Carruthers, L. M., Bednar, J., Woodcock, C. L., Hansen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linker+histones+stabilize+the+intrinsic+salt-dependent+folding+of+nucleosomal+arrays:+mechanistic+ramifications+for+higher-order+chromatin+folding.">Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding.</a> Biochemistry. 37 (42), 14776-14787 (1998).</li><li>Huynh, V. A. T., Robinson, P. J. J., Rhodes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+In+Vitro+Reconstitution+of+a+Defined+&amp;#34;30+nm&amp;#34;+Chromatin+Fibre+Containing+Stoichiometric+Amounts+of+the+Linker+Histone.">A Method for the In Vitro Reconstitution of a Defined &amp;#34;30 nm&amp;#34; Chromatin Fibre Containing Stoichiometric Amounts of the Linker Histone.</a> Journal of Molecular Biology. 345 (5), 957-968 (2005).</li><li>Dorigo, B., Schalch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+fiber+folding:+requirement+for+the+histone+H4+N-terminal+tail.">Chromatin fiber folding: requirement for the histone H4 N-terminal tail.</a> J. Mol. Biol. 2836 (03), 85-96 (2003).</li><li>Qian, R. L., Liu, Z. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+chromatin+folding+patterns+in+chicken+erythrocytes+by+atomic+force+microscopy+(AFM.">Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy (AFM.</a> Cell Research. 7 (2), 143-150 (1997).</li></ol>

יש המחברים אין ניגודי אינטרסים.

מערכי nucleosomal מודל הם כלי מאוד שימושי עבור המחקר במבחנה של מבנה הכרומטין ותפקוד. לדוגמא, הם היו בשימוש נרחב כדי ללמוד את המנגנון של עיבוי הכרומטין סיבים בפתרון 30-34, ועשו את זה ניתן להשיג מבנה צילום רנטגן של tetranucleosome 35. לאחרונה הם הוכיחו שימושיים בפענוח ה...

הגנום אוקריוטים לא קיים DNA עירום כ, אלא הם נדחסו ומאורגנים על ידי חלבונים מאוגדים. מתחמים אלה של ה-DNA וחלבונים ידועים כמו הכרומטין. היחידה החוזרת הבסיסית של הכרומטין היא הנוקלאוזום. הנוקלאוזום מורכב מoctamer היסטון ו146 זוגות בסיסים של DNA העטוף סביב octamer היסטון על פי 1.6 1. Octamer היסטון מורכב משני עותקים כל אחד מH2A ההיסטונים ליבה, H2B, H3 ו-H4. octamers היסטון ליבה שהם מרווחים שוב ושוב לאורך מולקולת הדנ&quot;א נקרא מערכי nucleosomal. המבנה המורחב של מערכי nucleosomal כבר מכונה סיבי 10 ננומטר או &quot;חרוזים על חוט&quot; מבנה ונמצא במבחנה בתנאים דלי מלח 2. סיבי 10 ננומטר הוא מסוגלים עיבוי לתוך מבנים מסדר גבוה יותר באמצעות דחיסה תוך מערך ו / או בין המערך oligomerization 2. יכולים להיגרם מבנים מסדר גבוה יותר אלה בנוכחותם של מלחים,או יכול להיות מושפע באמצעות מחייב של חלבונים אדריכליים הכרומטין למערך nucleosomal 3,4. רמות של דחיסת הכרומטין מתואמות באופן הפוך לשיעור של שעתוק ב5,6 vivo. המחקרים אחרונים מדגיש את החשיבות של הארגון המבני של הגנום בתהליכים כגון בידול, התפתחות הסרטן, ואחרים 7,8. השימוש במערכי nucleosomal ללמוד מבנה הכרומטין ותפקוד הפך נפוצים. כאן אנו מתארים שיטה להרכבה של מערכי nucleosomal מההיסטונים ליבת רקומביננטי ו-DNA מיצוב הנוקלאוזום. באמצעות DNA רקומביננטי בחוזר טנדם של רצפי מיצוב הנוקלאוזום מאפשר לכינון מחדש של מערכים המכילים nucleosomes במרווח קבוע. שניים מהרצפים יותר הפופולריים המיצוב הם רצף גן 5S rRNA ו9,10 &quot;601&quot; הרצף. רצף 601 נגזר מהניסויים Selex ומורדואר בחריפות עמדות nucleosomes מאשר רצף 5S 11. כתוצאה מכך, אורך ה-DNA מקשר של 601 המערכים הוא הומוגנית יותר. ה-DNA מיצוב הנוקלאוזום חוזר Tandemly מתקבלת על ידי סינון ג&#39;ל 4,12. ההיסטונים רקומביננטי הם מטוהרים מE. coli בתנאי denaturing 13. השימוש של ההיסטונים רקומביננטי מאפשר לשלוט בזהירות את הרכב היסטון של מערכי nucleosomal. לדוגמא, ליבת ההיסטונים נושאות מוטציות ספציפיות 14 או לאחר translational שינויים 15,16 יכול להיות תחליף ההיסטונים ליבה מסוג בר. ניסויי מהירות שקיעה לפקח על קצב השיקוע של מקרומולקולות בתמיסה תחת כוח הצנטריפוגלי הוחל 17. זה מניב מידע על הגודל וצורה של מקרומולקולות במדגם. ניסויי מהירות שקיעה הם אפוא כלי מתאים לחקר שינויי מדינת פתרון בסיבים הכרומטיןמבנה בשל עיבוי הכרומטין 18. חשוב מכך, יש צורך קודם כל להשתמש בניסויי מהירות שקיעה כצעד בקרת איכות בכינון מחדש מערך nucleosomal. אם ה-DNA וnucleosomes אינם משולבים ביחס טוחנת הראוי, המערכים עשויים להיות מתחת או עם ההיסטונים ליבה מעל הרווי. לפיכך, המידע שנצבר מניסויים מהירות שקיעה משמש כדי להבטיח כי ה-DNA הוא רווי כראוי עם nucleosomes. חשוב להשתמש בשיטות חלופיות כדי להעריך את הרוויה של DNA עם nucleosomes, במיוחד אם עובד עם תבנית ה-DNA uncharacterized בעבר. לכן, אנו גם מתארים שיטה לניתוח של מערכי nucleosomal באמצעות מיקרוסקופ כוח אטומי (AFM). AFM הוא טכניקה רבת עוצמה המאפשרת הדמיה של ההשפעות של מספר הפרמטרים, כגון הרמה של רוויה, השפעה של הנוכחות של וריאנטים היסטון או את ההשפעות של MgCl 2 19,20. AFM כבר גם appliאד ללמוד דינמיקת הנוקלאוזום באמצעות זמן לשגות הדמיה 21. במבחנה התאסף מערכי 12-mer nucleosomal הם נוחים במיוחד למחקרי AFM בטווח הגודל הנכון עבור ההדמיה AFM 22 כי הם שייכים. במחקר הנוכחי השתמשנו AFM של מערכי nucleosomal כמו בקרת איכות, כמו גם כאמצעי המאשר את הנתונים מAUC (&quot;לראות זה להאמין&quot;). בנוסף להדמיה פשוטה, AFM מאפשר מדידה של פרופילי גובה של דגימות כמדד נוסף.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(3-Aminopropyl)triethoxysilane</td>
 <td>Sigma-Aldrich </td>
 <td>A3648-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td>6-8 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>21-152-5</td>
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 <td>HiLoad Superdex 200 16/60 Column</td>
 <td>GE</td>
 <td>17-1069-01</td>
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 <td>Vivaspin 50 kDa MWCO Centrifugal Concentrator</td>
 <td>Sartorius</td>
 <td>VS2031</td>
 <td></td>
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 <td>12-14 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>08-667A</td>
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 <td>Illustra Sephacryl S-1000 Superfine</td>
 <td>GE</td>
 <td>17-0476-01 </td>
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 <td>XL-A/I Analytical Ultracentrifuge</td>
 <td>Beckman-Coulter</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning dna

octamers היסטון ליבה שהם מרווחים שוב ושוב לאורך מולקולת הדנ&quot;א נקרא מערכי nucleosomal. מערכי nucleosomal מתקבלים באחת משתי דרכים: טיהור מin vivo מקורות, או הכינון מחדש במבחנה מההיסטונים ליבת רקומביננטי ו-DNA מיצוב הנוקלאוזום חוזר tandemly. השיטה השנייה יש את היתרון של המאפשר הרכבה של מדים compositionally יותר ומערך nucleosomal ממוקם בדיוק. ניסויי מהירות שקיעה במידע תשואת ultracentrifuge אנליטיים בערך בגודל ובצורה של מקרומולקולות על ידי ניתוח בקצב שבו הם נודדים דרך פתרון מתחת לכוח הצנטריפוגלי. טכניקה זו, יחד עם מיקרוסקופ כוח אטומי, יכולה לשמש לבקרת איכות, על מנת להבטיח כי הרוב המכריע של תבניות DNA רווי בnucleosomes לאחר הכינון מחדש. כאן אנו מתארים את הפרוטוקולים הדרושים כדי לשקם את כמויות מיליגרם של אורך וcompositionally דמערכי nucleosomal efined מתאימים למחקרים ביוכימיים וbiophysical של מבנה הכרומטין ותפקוד.

כדי להמחיש את הפרוטוקול אנחנו מחדש מערכי nucleosomal מההיסטונים רקומביננטי Xenopus הליבה וה-DNA המורכב מבד בבד 12 207 חוזרים נ&quot;ב של רצף 601 מיצוב (601 207 x 12). אנחנו התאספנו ראשון octamers יליד מההיסטונים ליבת lyophilized ולאחר מכן מטוהרים octamers ידי FPLC באמצעות עמודת S200 (איור 1 א).<...

Watch this Scientific Journal Video about Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA at JoVE.com

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

שיטה מוצגת לכינון מחדש של מערכי nucleosomal מודל מההיסטונים ליבת רקומביננטי ו-DNA מיצוב הנוקלאוזום חוזר tandemly. אנו גם מתארים כיצד ניסויי מהירות שקיעה בultracentrifuge האנליטיות, ומיקרוסקופ כוח אטומי (AFM) משמשים לניטור לאחר הכינון מחדש את מידת הרוויה מערך nucleosomal.

הרכבה של nucleosomal מערכים מההיסטונים ליבת רקומביננטי והנוקלאוזום המיקום DNA

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

assembly-nucleosomal-arrays-from-recombinant-core-histones-nucleosome

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22.4K Views.  Colorado State University.  שיטה מוצגת לכינון מחדש של מערכי nucleosomal מודל מההיסטונים ליבת רקומביננטי ו-DNA מיצוב הנוקלאוזום חוזר tandemly. אנו גם מתארים כיצד ניסויי מהירות שקיעה בultracentrifuge האנליטיות, ומיקרוסקופ כוח אטומי (AFM) משמשים לניטור לאחר הכינון מחדש את מידת הרוויה מערך ...

Video: Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Isolation of Genomic DNA from Mouse Tails

בידוד של הדנ&quot;א הגנומי של זנבות עכבר

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

שילוב QD-סריג ו מיקרופלואידיקה לפקח Nanocomplex DNA הרכבה עצמית בזמן אמת

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

העצרת, טוען, ואת המערך של תא ultracentrifuge אנליטית לדוגמא

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

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 ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

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 expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

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 ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

עיצוב ושימוש במערכי Chemostat Multiplexed

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

הפקה וטיהור יעילה של רקומביננטי Murine Kindlin-3 מתאי חרקים לBiophysical לימודים

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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 ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

טיהור ללא chromatographic של פוליפפטידים דמויי אלסטין רקומביננטי וFusions עם פפטידים וחלבונים מן<em&gt; חיידקי Escherichia</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

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 ...