Les auteurs remercient Mme Jennifer M. Lommel et Tara N. Rognan pour l&#39;entretien des plantes d&#39;Arabidopsis. Ce travail a été soutenu par des fonds de démarrage de Saint Louis University et National Institutes of Health subventions 1R15GM086846-01 et 3R15GM086846-01S1 à W. Xiao.

I. croisement génétique 1. Amputer le parent femelle Afin de distinguer les allèles maternels et paternels en utilisant le polymorphisme de séquence d&#39;ADN, deux écotypes différents, par exemple-Britannique-0 (Col-0) et Ler, seront choisis en tant que parents féminin et masculin. Les plantes doivent être jeune et saine. On peut affaiblir le parent femelle en utilisant un microscope à dissection, grossissant visière, ou de l&#39;œil nu. Localisez étape-12 fleurs (Smyth et al., 1990) et enlever les fleurs ou siliques ci-dessus et en dessous d&#39;eux par clipsage de la base du pédoncule avec les ciseaux. Stériliser une pince en trempant la base de la pointe doucement dans un bécher d&#39;éthanol à 95%, ce qui supprime tout les grains de pollen sur la pince et de tuer le pollen ainsi. Soulever doucement dehors les bourgeons floraux à l&#39;aide des pinces, et retirez délicatement 4 sépales, 4 pétales, étamines et 6, en laissant le pistil nu et intact. Essayez d&#39;éviter d&#39;endommager le carpelles pendant ce processus. 2. Choisir le donneur de pollen et de réaliser la pollinisation Les donateurs meilleurs pollen sont anthesed fleurs ouvertes au stade 14 avec les pétales s&#39;étendant à angle de 90 ° au pistil (Smyth et al., 1990), dans lequel beaucoup de pollen est mue. Prenez la fleur à la base et juste au-dessus du pédoncule, qui va provoquer la fleur de répandre ouvert. La poussière du stigmate du pistil préparé avec l&#39;anthère. Après la pollinisation, la stigmatisation sera couvert avec le pollen jaune, qui peut être facilement observés sous un microscope. 3. Étiquetage de la croix Après la pollinisation tous les pistils émasculée sur une plante, nous marquons la croix avec une étiquette de bijoux et d&#39;information des parents masculins et féminins et la date. Placez une participation dans le sol près de l&#39;usine, utilisez une chaîne pour attacher la tige de l&#39;inflorescence au bûcher, et couvrir les pistils pollinisées avec un sac en plastique. II. Isolement des tissus endosperme chez Arabidopsis 1. Préparation des matériaux Nous obtenons habituellement des matériaux nécessaires avant la récolte de l&#39;endosperme et les tissus d&#39;embryon: un réservoir d&#39;azote liquide, azote liquide, un microscope à dissection, deux nouvelles paires de pointe fine pince (5 INOX TSF par Dumont biologie, Suisse.), Des lames de verre de microscope ( 3 &quot;x 1&quot; x 1.0 mm), une solution de pH 5,7 à 0,3 M de sorbitol et 5 mM de MES. 2. Collecte des siliques A 7 - ou 8-jour-après-pollinisation (DAP), les graines sont prêtes à être récoltées à la mi-à fin stade torpille de l&#39;embryogenèse et disséquées pour l&#39;endosperme et l&#39;embryon. Parfois, cela peut prendre 90 à 10 DAP si les fleurs sont trop jeunes émasculés. 3. Isoler l&#39;endosperme et l&#39;embryon Comme les graines d&#39;Arabidopsis sont minuscules, on utilise un microscope à dissection pour isoler l&#39;albumen et l&#39;embryon. Mettez une silique 8-DAP sous un microscope, utilisez une pince pour tenir le pédoncule silique et utilisez la pointe d&#39;une autre paire de pinces pour ouvrir le slide silique dans la marge, où fusionnent deux carpelles. Utilisez une paire de pinces pour ramasser une graine sur des solutions pH 5,7 de 0,3 M de sorbitol et 5 mM MES, faire une petite coupure à l&#39;extrémité micropyle à glisser l&#39;embryon, presser la fin uncut pour faire sortir l&#39;endosperme et l&#39;endosperme séparer de tégument (Kinoshita et al., 2004). Mettez l&#39;embryon et l&#39;endosperme dans des microtubes séparés dans l&#39;azote liquide. Continuer ainsi jusqu&#39;à ce que nous accumulons embryon ou l&#39;albumen de 10 à 15 siliques et stocker ensuite le tube dans un congélateur à -80 ° C. Dans certains cas, l&#39;albumen ne doit pas être séparé de tégument, c&#39;est à dire, on peut isoler l&#39;ARN total de l&#39;endosperme et le mélange de tégument pour l&#39;analyse des empreintes génétiques. III. Séquençage bisulfite 1. Réactifs nécessaires Bromure de cétyltriméthylammonium (CTAB) pour la préparation de l&#39;ADN génomique, des enzymes de restriction, 3 M NaOH (fraîchement faite), 6,42 M d&#39;urée / 4 de bisulfite de sodium M (2 métabisulfite de sodium M, Sigma-Aldrich, S9000, Na2S2O5 Poids Moléculaire: 190), 10 mM d&#39;hydroquinone, un kit de purification d&#39;ADN (DNA Promega Wizard système de nettoyage, Cat. # A7280), un tampon TE, 6,3 M de NaOH (fraîchement préparé), 10 M NH 4 OAc, 20 ug / uL ARNt, et de l&#39;éthanol à 100% . 2. Détails sur le protocole de traitement au bisulfite <ol><li> Isoler l&#39;endosperme ou l&#39;ADN génomique en utilisant une procédure d&#39;embryon CTAB (Rogers et Bendich, 1988). </li><li> Recueil de 100 ng - 2 pg d&#39;ADN génomique dans 20 uL à 100 uL volume total des enzymes de restriction qui coupent l&#39;extérieur de la région pour être analysés. Pour le promoteur MEA, nous utilisons Xhol, Ndel et PstI ou HindIII. </li><li> Dénaturer les enzymes de restriction par l&#39;ébullition de l&#39;ADN pour cinq minutes, puis étancher sur la glace. </li><li> Ajouter le volume 1 / 9 (2,2 pour 20 uL d&#39;ADN digérés uL) de 3 M NaOH et incuber à 37 ° C pendant 15 minutes. </li><li> Transférer la solution à un tube de PCR 250 uL. </li><li> Dissoudre 7,5 g d&#39;urée dans 10 ml d&#39;eau distillée stérile; Ajouter lentement 7,6 g de métabisulfite de sodium pendant 1-2 heures et de chauffage permet habituellement dissoudre; Ajuster le pH à 5 avec NaOH fraîchement préparé 10 M; Ajouter l&#39;eau distillée stérile à une finale volume à 20 ml. C&#39;est 6,24 M d&#39;urée / solution 4 M de bisulfite de sodium. </li><li> Ajouter 6,24 M d&#39;urée / solution 4 M de bisulfite de sodium à une concentration finale de 5,36 M et 3,44 M, respectivement (Paulin et al., 1998). Par exemple, ajouter 208 ul de 6,42 M d&#39;urée / solution 4 M de bisulfite de sodium à ce qui précède 22,2 uL d&#39;ADN génomique dénaturé (Xiao et al., 2003). </li><li> Ajouter 10 mM d&#39;hydroquinone à l&#39;ADN à une concentration finale de 0,5 mM (12 uL de 20 uL de digestion). </li><li> Conduite traitement au bisulfite dans une machine à PCR: 30 cycles de 55 ° C pendant 15 minutes et 95 ° C pendant 30 secondes. </li><li> Dessaler l&#39;ADN au bisulfite traitées en utilisant l&#39;ADN Assistant Nettoyage du système de Promega et le suivi du protocole (Jacobsen et al., 2000). </li><li> Mesurez le volume exact de TE récupéré de la colonne après dessalage et ajouter 6,3 M NaOH à une concentration finale de 0,3 M. Incuber à 37 ° C pendant 15 minutes. </li><li> Ajouter 10 M NH 4 OAc (pH 7,0) à une concentration finale de 3 M, 2 pl de 20 ug / uL ARNt, et 3 volumes d&#39;éthanol 100%, puis mélanger. Centrifuger pendant 15 minutes à 14000 rpm. </li><li> Laver le culot une fois avec 70% d&#39;éthanol, une centrifugeuse ne court, et éliminer l&#39;éthanol supplémentaire. </li><li> Culot sec dans une speedvac pendant 5-10 minutes et remettre en suspension dans 25 à 100 pi de tampon TE selon le montant de départ de l&#39;ADN. Le bisulfite de sodium-traitée ADN est maintenant prêt pour l&#39;analyse PCR. </li></ol> 3. L&#39;amplification par PCR <ol><li> Depuis cytosines non méthylées sont converties en uracile, il est difficile pour amplifier un fragment volumineux à l&#39;aide de l&#39;ADN au bisulfite traitée comme un modèle. Ainsi, nous avons l&#39;habitude concevoir des amorces pour amplifier un produit pas plus de 500 pb. Pour la séquence du promoteur de 4 kb MEA, nous avons conçu de nombreux ensembles d&#39;amorces et amplifié 14 fragments qui se chevauchent pour couvrir toute la région (Xiao et al., 2003). </li><li> Pour la séquence du haut-brin, dans la conception d&#39;une amorce de l&#39;avant, I) choisir un G (guanine) région riche afin d&#39;avoir une température plus élevée de recuit sans supplément nucléotides de long dans les amorces; II) le changement C (cytosine) à Y ( pyrimidine) à CG et contextes GNC et changer le C restant à T (thymine). Dans la conception d&#39;une amorce antisens, I) choisissez une région C-riche; II) le changement G à R (purines) au CG et contextes GNC et le changement du G restant à A (adénine). </li><li> Pour séquencer le bas-brin, à concevoir une amorce sens, I) choisissez une région C-riche; II) le changement G à R à CG et contextes GNC et le changement du G restant à A. Dans la conception d&#39;une amorce antisens, I) choisissent une région riche en G; II) le changement C à Y (pyrimidine) à CG et contextes GNC et changer le C restant à T. </li><li> Nous utilisons habituellement 1-2 uL de la bisulfite de sodium-traitée ADN comme un modèle pour chaque amplification par PCR (Xiao et al., 2003). Le produit de PCR doit être analysé par électrophorèse sur gel pour confirmer la bonne taille du fragment, puis d&#39;être purifiés sur gel et cloné dans le clonage TOPO TA pCR2.1 vecteur (Invitrogen) comme un insert. Une seule colonie est ramassé et mis en culture; ADN plasmidique extrait et envoyé pour le séquençage. </li></ol> 4. L&#39;analyse des séquences Le principe du séquençage au bisulfite est que la cytosine non méthylé sera convertie en uracile due à la désamination hydrolytique par une forte concentration de bisulfite de sodium à pH 5,0, qui sera amplifié comme la thymine dans le produit PCR, tandis que la 5-méthyl cytosine ne sera pas modifié par le bisulfite de sodium et restent à la cytosine après amplification par PCR (Clark et al, 1994;. Frommer et al, 1992).. Après l&#39;obtention du résultat de séquençage, on le compare avec le modèle brin spécifique qui est utilisé pour l&#39;amplification par PCR. Si un résidu cytosine dans le modèle se lit comme une thymine dans le résultat de séquençage, il indique que la cytosine n&#39;est pas méthylé. Si un résidu cytosine dans le modèle reste une cytosine dans le séquençage, cela signifie que la cytosine est méthylée. IV. Les résultats représentatifs <img src="/files/ftp_upload/2327/2327fig1.jpg" alt="Figure 1 /" /> Figure 1.

<ol>
	<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., Sriharsa Pradhan, S., Nelson, S. F., Pellegrini, M., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+bisulphite+sequencing+of+the+Arabidopsis+genome+reveals+DNA+methylation+patterning.">Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.</a> Nature. 452, 215-219 (2008).</li><li>Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc Natl Acad Sci USA. 89, 1827-1831 (1992).</li><li>Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B., Fischer, R. L. D. E. M. E. T. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+glycosylase+establishes+MEDEA+polycomb+gene+self-imprinting+by+allele-specific+demethylation.">DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.</a> Cell. 124, 495-506 (2006).</li><li>Henderson, I. R., Chan, S. R., Cao, X., Johnson, L., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+sodium+bisulfite+sequencing+in+plants.">Accurate sodium bisulfite sequencing in plants.</a> Epigenetics. 5, 47-49 (2010).</li><li>Hsieh, T. F., Ibarra, C. A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R. L., Zilberman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+demethylation+of+Arabidopsis+endosperm.">Genome-wide demethylation of Arabidopsis endosperm.</a> Science. 324, 1451-1454 (2009).</li><li>Jacobsen, S. E., Sakai, H., Finnegan, E. J., Cao, X., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+hypermethylation+of+flower-specific+genes+in+Arabidopsis.">Ectopic hypermethylation of flower-specific genes in Arabidopsis.</a> Curr. Biol. 10, 179-186 (2000).</li><li>Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., Kakutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-way+control+of+FWA+imprinting+in+Arabidopsis+endosperm+by+DNA+methylation.">One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation.</a> Science. 303, 521-523 (2004).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+integrated+single-base+resolution+maps+of+the+epigenome+in+Arabidopsis.">Highly integrated single-base resolution maps of the epigenome in Arabidopsis.</a> Cell. 133, 523-536 (2008).</li><li>Lister, R., Pelizzola, M., Dowen, R. H., Hawkins, R. D., Hon, G., Tonti-Filippini, J., Nery, J. R., Lee, L., Ye, Z., Ngo, Q. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+methylomes+at+base+resolution+show+widespread+epigenomic+differences.">Human DNA methylomes at base resolution show widespread epigenomic differences.</a> Nature. 462, 315-322 (2009).</li><li>Paulin, R., Grigg, G. W., Davey, M. W., Piper, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urea+improves+efficiency+of+bisulfite-mediated+sequencing+of+5'-+methylcytosine+in+genomic+DNA.">Urea improves efficiency of bisulfite-mediated sequencing of 5'- methylcytosine in genomic DNA.</a> Nucl. Acids Res. 26, 5009-5010 (1998).</li><li>Rogers, S. O., &amp;amp, B. e. n. d. i. c. h., J, A. <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+plant+tissues.">Extraction of DNA from plant tissues.</a> Plant Molecular Biology Manual. A6, 1-10 (1988).</li><li>Smyth, D. R., Bowman, J. L., Elliot, M., Meyerowitz, E. 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Il est relativement facile de séparer embryon à partir de l&#39;endosperme et tégument, mais il est fastidieux de séparer l&#39;endosperme du tégument, en particulier pour les semences à un stade précoce ou à mi-torpilles de l&#39;embryogenèse. Depuis tégument ne contribue qu&#39;à une très petite quantité de tissu, pour certains gènes, par exemple, la MEA et FWA, nous n&#39;avons pas de séparer l&#39;endosperme du tégument. Cela signifie que nous pouvons isoler les ARN à partir d&#39;...

Fournitures <ul><li> Microscope à dissection </li><li> Ciseaux </li><li> Pince pointe fine </li><li> Tag bijoux </li><li> Enjeux des végétaux </li><li> String ou les attaches </li><li> 4 &quot;X 2&quot; X 8 sacs en polyéthylène &quot; </li><li> 3 &quot;X 1&quot; X 1,0 mm Lames de microscope </li><li> Azote liquide </li><li> Conteneurs d&#39;azote liquide </li><li> Bloc de chaleur </li><li> Tubes PCR </li><li> Thermocycleur </li><li> Microtubes à centrifuger </li><li> Microcentrifugeuse </li><li> Installation électrophorèse sur gel </li><li> Arabidopsis thaliana-0-Britannique Plantes </li><li> Arabidopsis thaliana Landsberg erecta Plantes </li></ul> Réactifs <ul><li> L&#39;éthanol à 70% </li><li> L&#39;éthanol à 95% </li><li> L&#39;éthanol à 100% </li><li> 0,3 M de sorbitol et 5 mM MES pH 5,7 </li><li> Bromure de cétyltriméthylammonium (CTAB) </li><li> L&#39;éthanol à 100% </li><li> Cholorform </li><li> Les enzymes de restriction </li><li> 3 M NaOH </li><li> 6,3 M de NaOH </li><li> 6,24 M d&#39;urée / 4 de bisulfite de sodium M </li><li> Distillée stérile H 2 O </li><li> 10 mM d&#39;hydroquinone </li><li> Assistant DNA Clean-Up System (Promega) </li><li> 10 M NH 4 OAc </li><li> 20 pg / uL ARNt </li><li> Tampon TE </li><li> Le kit de clonage TOPO TA (Invitrogen) </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana est un organisme excellent modèle pour étudier les mécanismes épigénétiques. Une des raisons est le mutant nul de perte de fonction de l&#39;ADN méthyltransférases est viable, offrant ainsi un système à étudier comment la perte de méthylation de l&#39;ADN dans un génome influe sur la croissance et le développement. Impression se réfère à l&#39;expression différentielle des allèles maternels et paternels et joue un rôle important dans le développement de la reproduction dans les deux mammifères et les plantes. Méthylation de l&#39;ADN est essentielle pour déterminer si les allèles maternels ou paternels d&#39;un gène est exprimé ou imprimé au silence. Dans les plantes à fleurs, il ya un événement double fécondation de la reproduction: un spermatozoïde fertilise l&#39;ovule pour former l&#39;embryon et un spermatozoïde fusionne avec la seconde cellule centrale pour donner naissance à l&#39;albumen. Endosperme est le tissu où se produit l&#39;empreinte dans les plantes. MEDEA, un domaine SET gènes du groupe Polycomb et FWA, un facteur de transcription régulant la floraison, sont les deux premiers gènes soumis à empreinte montré dans l&#39;endosperme et de leur expression est contrôlée par méthylation de l&#39;ADN et la déméthylation de plantes. Afin de déterminer le statut de l&#39;empreinte d&#39;un gène et le modèle de méthylation dans l&#39;albumen, nous devons être en mesure d&#39;isoler l&#39;albumen premier. Depuis semence est petite chez Arabidopsis, il reste difficile d&#39;isoler l&#39;albumen Arabidopsis et examiner sa méthylation. Dans ce protocole vidéo, nous présentons la façon de mener un croisement génétique, afin d&#39;isoler albumen de graines, et de déterminer le statut de méthylation par séquençage au bisulfite.

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

L&#39;imprinting est un phénomène dans l&#39;usine et la reproduction des mammifères. Méthylation de l&#39;ADN joue un rôle important dans les mécanismes de l&#39;empreinte. Isoler l&#39;endosperme et la détermination du statut de méthylation de gènes soumis à empreinte dans les<em&gt; Arabidopsis</em&gt; Il peut être difficile. Dans ce protocole, nous décrivons comment isoler l&#39;albumen et de déterminer la méthylation par séquençage au bisulfite.

Détermination de méthylation de l&#39;ADN des gènes soumis à empreinte dans les Arabidopsis Endosperme

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

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Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.3K vues.  Saint Louis University. L'imprinting est un phénomène dans l'usine et la reproduction des mammifères. Méthylation de l'ADN joue un rôle important dans les mécanismes de l'empreinte. Isoler l'endosperme et la détermination du statut de méthylation de gènes soumis à empreinte dans les Arabidopsis Il peut être difficile. Dans ce protocole, nous décrivons comment isoler l'albumen et de déterminer la méthylation par séquençage au bisulfite.

Vidéo: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Méthylation de l&#39;ADN: bisulfite de modification et d&#39;analyse

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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Biologie

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: détection d&#39;altérations numériques et structurelles dans les 24 chromosomes de l&#39;homme en utilisant simultanément un dosage FISH Novel OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Un tégument Literie dosage à explorer génétiquement<em&gt; In Vitro</em&gt; Comment la germination des contrôles endosperme des semences dans<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

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.

Imagerie en temps réel de l&#39;unité d&#39;ingénierie des ARN transcrits dans des cellules vivantes utilisant Ratiométrique bimoléculaires 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 ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro test de Méthylation à étudier des protéines méthylation de l&#39;arginine

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Enhanced réduit Représentation bisulfite séquençage pour l&#39;évaluation de méthylation de l&#39;ADN à la résolution de paires de base

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Corréler spécifique du gène ADN changements méthylation avec l&#39;expression et l&#39;activité transcriptionnelle des astrocytes<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Ciblé méthylation de l&#39;ADN Analyse par séquençage de nouvelle génération

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprécipitation de la chromatine test pour l&#39;identification de Arabidopsis interactions protéine-ADN<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Résolution Quantification haut de Crystalline Cellulose Accumulation dans<em&gt; Arabidopsis</em&gt; Roots pour surveiller cellulaire spécifique d&#39;un tissu mural Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...