Die Autoren danken Frau Jennifer M. Lommel und Tara N. Rognan für die Wartung von Arabidopsis-Pflanzen. Diese Arbeit wurde durch eine Anschubfinanzierung von Saint Louis University und National Institutes of Health gewährt 1R15GM086846-01 und 3R15GM086846-01S1 zu W. Xiao unterstützt.

I. Genetische Crossing 1. Eine Schwächung des weiblichen Elternteils Um die mütterlichen und väterlichen Allele durch DNA-Sequenz-Polymorphismus, zwei verschiedene Ökotypen, zB Columbia-0 (Col-0) und Ler, wird als weiblich und männlich Eltern gewählt werden, zu unterscheiden. Die Pflanzen sollten jung und gesund. Man kann das weibliche Elternteil mit einem Binokular entmannen, Vergrößerungsspiegel Visier, oder mit bloßem Auge. Suchen Bühne-12 Blumen (Smyth et al., 1990) und entfernen Sie alle Blumen oder Schoten über und unter ihnen durch Abschneiden der Basis des Stiels mit der Schere. Sterilisieren Pinzette durch Eintauchen der Basis der Spitze vorsichtig in ein Becherglas mit 95% Ethanol, die alle Pollenkörner an der Zange wird zu entfernen und zu töten den Pollen als auch. Hebeln auseinander die Blütenknospen mit einer Pinzette und sanft Entfernen 4 Kelchblätter, 4 Blütenblätter, Staubblätter und 6, so dass die Stempel kahl und intakt. Versuchen Sie, um eine Beschädigung der Fruchtblätter während dieses Prozesses zu vermeiden. 2. Die Auswahl der Pollenspender und Durchführung Bestäubung Die besten Pollen Geber offene Blüten im Stadium 14 anthesed mit den Blütenblättern Erweiterung in einem 90 °-Winkel zu den Stempel (Smyth et al., 1990), in denen eine Menge von Pollen ist zu vergießen. Besorgen Sie sich die Blume an der Basis und knapp oberhalb des Stiels, die bewirkt, dass die Blume zu gespreizt. Staub das Stigma der vorbereiteten Stempel mit der Anthere. Nach der Bestäubung, wird das Stigma, mit dem gelben Pollen, die leicht unter dem Mikroskop beobachtet werden kann abgedeckt werden. 3. Labeling Kreuz Nach Bestäubung alle entmannt Stempel auf einer Pflanze, markieren wir das Kreuz mit einem Schmuck-tag und Informationen von weiblichen und männlichen Eltern und Datum. Legen Sie einen Pfahl in den Boden in der Nähe der Anlage, verwenden Sie einen String an den Stiel der Blüte auf den Scheiterhaufen binden, und decken den bestäubten Stempeln mit einer Plastiktüte. II. Isolierung von Endosperm Gewebe in Arabidopsis 1. Vorbereitung von Materialien Wir Regel erhalten notwendigen Materialien bereit vor der Ernte das Endosperm und Embryo Gewebe: eine flüssige Stickstoff-Tank, flüssiger Stickstoff, einem Binokular, zwei neue Paar feine Spitze Pinzette (5 INOX FST von Dumont Biologie, Schweiz.), Mikroskop Objektträger ( 3 &quot;X 1&quot; x 1,0 mm), einem pH 5,7 Lösung von 0,3 M Sorbit und 5 mM MES. 2. Sammeln Schoten Am 7 - oder 8-Tage-after-Bestäubung (DAP), sind die Samen bereit, an der mittleren bis späten Stadium der Embryogenese Torpedo geerntet werden und seziert für Endosperm und Embryo. Manchmal kann es dauern 9-10 DAP, wenn die entmannt Blumen zu jung sind. 3. Isolating Endosperm und Embryo Da Arabidopsis Samen sind winzig, verwenden wir ein Binokular auf Endosperm und Embryo zu isolieren. Setzen Sie ein 8-DAP Schote unter dem Mikroskop, verwenden Sie eine Pinzette, um die Schote Stiel halten und mit der Spitze eines anderen Zange zum Öffnen der Schote an den Rand, wo zwei Fruchtblätter Sicherung schieben. Verwenden Sie eine Pinzette zu holen einen Samen auf pH 5,7 Lösung von 0,3 M Sorbit und 5 mM MES, machen einen kleinen Schnitt an der Mikropyle Ende zu gleiten Embryo, drücken Sie die uncut Ende zu verdrängen Endosperm und separate Endosperm von Samenschale (Kinoshita et al., 2004). Setzen Sie den Embryo und Endosperm in separate Röhrchen in flüssigem Stickstoff. Setzen Sie diesen, bis wir Embryo oder Endosperm von 10 bis 15 Schoten sammeln und speichern Sie dann das Rohr in einem -80 ° C Gefrierschrank. In einigen Fällen, Endosperm nicht aus Samenschale, dh getrennt werden, kann man Gesamt-RNA aus dem Endosperm und Samenschale Mischung für die Gen-Imprinting-Analyse zu isolieren. III. Bisulfit-Sequenzierung 1. Benötigte Reagenzien Cetyltrimethylammoniumbromid (CTAB) zur Herstellung von genomischer DNA, Restriktionsenzyme, 3 M NaOH (frisch zubereitet), 6,42 M Harnstoff / 4 M Natriumbisulfit (2 M Natriummetabisulfit, Sigma-Aldrich, S9000, Na2S2O5, Molekulargewicht: 190), 10 mM Hydrochinon, ein DNA Purification Kit (Promega Wizard DNA Clean-up-System, Cat. # A7280), TE-Puffer, 6,3 M NaOH (frisch), 10 M NH 4 OAc, 20 ug / ul tRNA, und 100% Ethanol . 2. Details der Bisulfit-Behandlung-Protokoll <ol><li> Isolieren Sie genomische Endosperm oder der Embryo DNA mit einer CTAB-Verfahren (Rogers und Bendich, 1988). </li><li> Digest 100 ng - 2 ug genomische DNA in 20 ul bis 100 ul Gesamtvolumen mit Restriktionsenzymen, die außerhalb der Region analysiert werden abgeschnitten. Für die MEA-Promotor, verwenden wir XhoI, NdeI und PstI oder HindIII. </li><li> Denaturieren die Restriktionsenzyme durch Kochen der DNA für 5 Minuten und dann löschen auf dem Eis. </li><li> Add 1 / 9 Volumen (2,2 ul für 20 ul verdaute DNA) von 3 M NaOH und Inkubation bei 37 ° C für 15 Minuten. </li><li> Übertragen Sie die Lösung für ein 250 ul PCR-Röhrchen. </li><li> Lösen von 7,5 g Harnstoff in 10 ml sterilem destilliertem Wasser; Langsam 7,6 g Natriummetabisulfit über 1-2 Stunden und Heizung hilft in der Regel lösen; pH-Wert auf 5 mit frisch zubereiteten 10 M NaOH; Add sterilem destilliertem Wasser zu einer endgültigen Volumen auf 20 mL. Dies ist 6.24 M Harnstoff / 4 M Natriumbisulfit-Lösung. </li><li> Add 6,24 m Harnstoff / 4 M Natriumbisulfit-Lösung auf eine Endkonzentration von 5,36 M und 3,44 M bzw. (Paulin et al., 1998). Fügen Sie z. B. 208 ul von 6,42 M Harnstoff / 4 M Natriumbisulfit Lösung für das obige 22,2 ul denaturierter genomischer DNA (Xiao et al., 2003). </li><li> Add 10 mM Hydrochinon, um die DNA in einer Endkonzentration von 0,5 mM (12 ul für 20 ul Verdauung). </li><li> Conduct Bisulfit-Behandlung in einer PCR-Maschine: 30 Zyklen von 55 ° C für 15 Minuten und 95 ° C für 30 Sekunden. </li><li> Desalt der Bisulfit-behandelten DNA mit Hilfe des Assistenten DNA Clean-Up-System von Promega und Follow-up des Protokolls (Jacobsen et al., 2000). </li><li> Messen Sie die exakte Volumen TE aus der Kolonne nach Entsalzung zurückgewonnen und fügen 6.3 M NaOH bis zu einer Endkonzentration von 0,3 M. Bei 37 ° C für 15 Minuten. </li><li> Add 10 M NH 4 OAc (pH 7,0) bis zu einer Endkonzentration von 3 M, 2 ul von 20 ug / ul tRNA und 3 Volumen 100% Ethanol und dann mischen. Zentrifuge für 15 Minuten bei 14.000 Umdrehungen pro Minute. </li><li> Waschen des Pellets einmal mit 70% Ethanol, nicht eine kurze Zentrifuge, und entfernen Sie zusätzliche Ethanol. </li><li> Dry Pellet in einem Speedvac für 5-10 Minuten und resuspendieren in 25 bis 100 ul TE-Puffer je nach Ausgangspunkt der DNA-Menge. Die Natrium-Bisulfit-behandelten DNA ist nun bereit für die PCR-Analyse. </li></ol> 3. PCR-Amplifikation <ol><li> Da nicht methylierter Cytosin in Uracil umgewandelt werden, ist es schwierig, ein großes Fragment mit der Bisulfit-behandelten DNA als Vorlage zu verstärken. So haben wir in der Regel Design-Primer, um ein Produkt nicht länger als 500 bp zu verstärken. Um Abfolge der 4-KB-MEA-Promotor, entwickelten wir viele Sätze von Primer und verstärkt 14 überlappende Fragmente auf die gesamte Region abdecken (Xiao et al., 2003). </li><li> Um Reihenfolge der Top-Strang, bei der Gestaltung eines Forward-Primer, I) wählen Sie einen G (Guanin)-reiche Region, um eine höhere Glühtemperatur ohne extra lang Nukleotide der Primer haben; II) ändern C (Cytosin) bis Y ( Pyrimidin) bei CG-und CNG-Kontexten und ändern Sie die restlichen C bis T (Thymin). In der Gestaltung eines Reverse-Primer, I) wählen Sie einen C-reiche Region; II) ändern G bis R (Purin) bei CG-und CNG-Kontexten und ändern Sie die restlichen G zu A (Adenin). </li><li> Um Abfolge der unteren Strang, bei der Gestaltung eines Forward-Primer, I) wählen Sie einen C-reiche Region; II) ändern G bis R am CG-und CNG-Kontexten und ändern Sie die übrigen G zu A. In der Gestaltung eines Reverse-Primer, I) wählen eine G-reiche Region; II) ändern C bis Y (Pyrimidin) bei CG-und CNG-Kontexten und ändern Sie die restlichen C bis T. </li><li> Wir in der Regel Verwenden Sie 1-2 ul der Natrium-Bisulfit-behandelten DNA als Vorlage für jedes PCR-Amplifikation (Xiao et al., 2003). Das PCR-Produkt muss durch Gel-Elektrophorese analysiert werden, um die richtige Größe des Fragments zu bestätigen, dann Gel gereinigt und kloniert in den TOPO TA-Klonierungsvektor pCR2.1 (Invitrogen) als Insert. Eine einzelne Kolonie wird aufgegriffen und kultiviert; Plasmid-DNA extrahiert und für die Sequenzierung. </li></ol> 4. Die Sequenzanalyse Das Prinzip der Bisulfit-Sequenzierung ist, dass methylierte Cytosin wird in Uracil durch hydrolytische Desaminierung durch eine hohe Konzentration von Natriumbisulfit bei pH 5,0, die als Thymin in PCR-Produkt wird verstärkt umgesetzt werden, während 5-Methylcytosin wird nicht durch Natriumbisulfit geändert werden und bleiben bis Cytosin nach PCR-Amplifikation (Clark et al, 1994;. Frommer et al, 1992.). Nach Erhalt der Sequenzierung durch, vergleichen wir sie mit dem Strang-spezifische Vorlage, die für die PCR-Amplifikation verwendet wird. Wenn ein Cytosinrest in der Vorlage liest sich wie ein Thymin in der Sequenzierung Ergebnis bedeutet dies, dass das Cytosin nicht methyliert ist. Wenn ein Cytosinrest in der Vorlage bleibt ein Cytosin in der Sequenzierung, bedeutet dies, dass das Cytosin methyliert ist. IV. Repräsentative Ergebnisse <img src="/files/ftp_upload/2327/2327fig1.jpg" alt="Abbildung 1 /" /> Abbildung 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. 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Es ist relativ leicht zu Embryo aus Endosperm und Samenschale getrennt, aber es ist mühsam, Endosperm von Samenschale trennen, vor allem für Saatgut auf Anfang oder Mitte-Torpedo Stadium der Embryogenese. Da Samenschale trägt nur eine sehr kleine Menge an Gewebe, für einige Gene, z. B., MEA und FWA, haben wir nicht zu Endosperm von Samenschale zu trennen. Das heißt, wir können RNAs aus einer Mischung von Endosperm und Samenschale Gewebe zu isolieren, zu überprüfen Ausdruck des mütterlichen und...

Vorräte <ul><li> Präpariermikroskop </li><li> Schere </li><li> Feine Spitze Pinzette </li><li> Schmuck Tag </li><li> Werk Stakes </li><li> String oder Twist-Ties </li><li> 4 &quot;X 2&quot; X 8 &quot;Polyethylen-Beutel </li><li> 3 &quot;X 1&quot; x 1,0 mm Mikropräparate </li><li> Flüssiger Stickstoff </li><li> Flüssigstickstoffbehältern </li><li> Heizblock </li><li> PCR Tubes </li><li> Thermocycler </li><li> Mikrozentrifugenröhrchen </li><li> Microcentrifuge </li><li> Gel-Elektrophorese-Anlage </li><li> Arabidopsis thaliana Columbia-0 Pflanzen </li><li> Arabidopsis thaliana Landsberg erecta Pflanzen </li></ul> Reagenzien <ul><li> 70% Ethanol </li><li> 95% Ethanol </li><li> 100% Ethanol </li><li> 0,3 M Sorbitol und 5 mM MES-pH 5,7 </li><li> Cetyltrimethylammoniumbromid (CTAB) </li><li> 100% Ethanol </li><li> Cholorform </li><li> Restriktionsenzyme </li><li> 3 M NaOH </li><li> 6.3 M NaOH </li><li> 6,24 m Urea / 4 M Natriumbisulfit </li><li> Sterilem destilliertem H 2 O </li><li> 10 mM Hydrochinon </li><li> Wizard DNA Clean-Up System (Promega) </li><li> 10 M NH 4 OAc </li><li> 20 ug / ul tRNA </li><li> TE-Puffer </li><li> Die TOPO TA Cloning Kit (Invitrogen) </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana ist ein hervorragendes Modell-Organismus für die Untersuchung epigenetischen Mechanismen. Einer der Gründe ist der Verlust-of-function Nullmutante von DNA-Methyltransferasen lebensfähig ist, so dass ein System zu untersuchen, wie Verlust von DNA-Methylierung in einem Genom beeinflusst Wachstum und Entwicklung. Imprinting bezeichnet differentielle Expression von mütterlichen und väterlichen Allelen und spielt eine wichtige Rolle bei der Fortpflanzung Entwicklung in beiden Säugetier-und Pflanzenarten. DNA-Methylierung ist entscheidend dafür, ob die mütterliche oder väterliche Allele eines aufgedruckten Gen exprimiert wird oder zum Schweigen gebracht. In Blütenpflanzen gibt es eine doppelte Befruchtung bei der Reproduktion: eine Samenzelle befruchtet die Eizelle zum Embryo und einem zweiten Spermien Sicherungen mit der zentralen Zelle zu steigen, um Endosperm Form geben. Endosperm ist das Gewebe, in dem Imprinting tritt in Pflanzen. MEDEA, ein SET-Domäne Polycomb Gruppe Gen und FWA, ein Transkriptionsfaktor Regulierung der Blüte sind die ersten beiden Gene gezeigt, dass in Endosperm und ihren Ausdruck geprägt wird durch DNA-Methylierung und Demethylierung in kontrollierten Pflanzen. Um Imprinting-Status eines Gens und Methylierungsmuster in Endosperm zu bestimmen, müssen wir in der Lage sein Endosperm zunächst zu isolieren. Da Samen ist winzig in Arabidopsis, bleibt es schwierig, Arabidopsis Endosperm isolieren und untersuchen ihre Methylierung. In diesem Video-Protokoll, berichten wir, wie man eine genetische Kreuz zu führen, um Endospermgewebe aus Samen zu isolieren und den Methylierungsstatus von Bisulfit-Sequenzierung zu ermitteln.

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

Imprinting ist ein Phänomen in Pflanzen-und Säugetier Reproduktion. DNA-Methylierung spielt eine wichtige Rolle in Mechanismen der Prägung. Isolating Endosperm und Bestimmung Methylierungsstatus von geprägten Genen in<em&gt; Arabidopsis</em&gt; Kann schwierig sein. In diesem Protokoll, beschreiben wir, wie Endosperm isolieren und zu bestimmen Methylierung durch Bisulfit-Sequenzierung.

Bestimmung der DNA-Methylierung von geprägten Genen in Arabidopsis Endosperm

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

determination-dna-methylation-imprinted-genes-arabidopsis

Research

JoVE Journal

Biology

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.4K Aufrufe.  Saint Louis University. Imprinting ist ein Phänomen in Pflanzen-und Säugetier Reproduktion. DNA-Methylierung spielt eine wichtige Rolle in Mechanismen der Prägung. Isolating Endosperm und Bestimmung Methylierungsstatus von geprägten Genen in Arabidopsis Kann schwierig sein. In diesem Protokoll, beschreiben wir, wie Endosperm isolieren und zu bestimmen Methylierung durch Bisulfit-Sequenzierung.

Serie: 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.

DNA-Methylierung: Bisulfit Modifikation und Analyse

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

Forschung

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: Erkennung von numerischen und strukturellen Veränderungen in alle 24 menschlichen Chromosomen gleichzeitig unter Verwendung eines neuartigen OctoChrome FISH-Test

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.

Ein Mantel Bettwäsche Seed-Test zur Genetisch erkunden<em&gt; In-vitro-</em&gt; Wie das Endosperm Kontrollen Keimung der Samen in<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.

Echtzeit-Bildgebung von Einzel Engineered RNA-Transkripte in lebenden Zellen Mit Ratiometrische Bimolekulare 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 ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enrichment durch eine Transposase-basierte Technologie für NGS-Analyse der gesamten Sequenz von<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt; Und 9 Gene in der DNA-Reparatur beteiligt Schäden

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

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 zur Methylierung Protein-Arginin-Methylierung Studieren

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.

Verbesserte Reduzierte Darstellung Bisulfit-Sequenzierung für die Bewertung der DNA-Methylierung bei Basenpaar Auflösung

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.

Korrelieren von Gen-spezifischen DNA-Methylierungsänderungen mit Expression und Transkriptionsaktivität Astrozytäre<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.

Gezielte DNA Methylierungsanalyse von 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 ...

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.

Chromatinimmunpräzipitation Assay zur Identifizierung von Arabidopsis-Protein-DNA-Wechselwirkungen<em&gt; In-vivo-</em

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