Os autores agradecem a Sra. Jennifer M. Lommel e Tara N. Rognan para a manutenção de plantas de Arabidopsis. Este trabalho foi financiado por fundos start-up de Saint Louis University e dos Institutos Nacionais de Saúde concede 1R15GM086846-01 e 3R15GM086846-01S1 para W. Xiao.

I. Genética Travessia 1. Castrar o pai do sexo feminino A fim de distinguir os alelos maternos e paternos, usando polimorfismo de DNA seqüência, dois ecótipos diferentes, por exemplo, Columbia-0 (Col-0) e Ler, serão escolhidos como pais feminino e masculino. As plantas devem ser jovens e saudáveis. Pode-se castrar o pai do sexo feminino, usando um microscópio de dissecação, ampliando viseira, ou olho nu. Localize estágio-12 flores (Smyth et al., 1990) e remover qualquer flores ou siliques acima e abaixo deles pelo recorte da base do pedicelo com a tesoura. Esterilizar fórceps por imersão a base da ponta suavemente para um copo de etanol a 95%, o que irá remover quaisquer grãos de pólen sobre o forceps e matar o pólen também. Force suavemente para além dos botões florais utilizando fórceps, e remova cuidadosamente 4 sépalas, 4 pétalas e 6 estames, deixando o pistilo nua e intacta. Tente evitar danos ao carpelos durante este processo. 2. Escolher o doador de pólen e realizar a polinização Os doadores de pólen são melhores anthesed flores abertas na fase 14 com as pétalas estendendo em um ângulo de 90 ° para o pistilo (Smyth et al., 1990), em que uma grande quantidade de pólen é derramamento. Pegue a flor na base e um pouco acima do pedicelo, que fará com que a flor a espalhar aberto. Poeira o estigma do pistilo preparado com a antera. Após a polinização, o estigma será coberto com o pólen amarelo, que pode ser facilmente observado sob um microscópio. 3. Rotulagem da cruz Depois de polinizadores todos os pistilos emasculados em uma planta, nós rotulamos a cruz com uma etiqueta de jóias e informações dos pais do sexo feminino e masculino e data. Coloque uma estaca no solo próximo à planta, use uma corda para amarrar a haste da inflorescência para a fogueira, e cobrem os pistilos polinizados com um saco plástico. II. Isolamento de tecidos endosperma em Arabidopsis 1. Preparando materiais Costumamos receber materiais necessários pronto antes da colheita do endosperma e dos tecidos do embrião: um tanque de nitrogênio líquido, nitrogênio líquido, um microscópio de dissecação, dois novos pares de fina ponta pinças (5 INOX FST por Dumont biologia, na Suíça.), Lâminas de vidro de microscópio ( 3 &quot;X 1&quot; X 1.0 mm), um pH 5,7 solução de 0,3 M sorbitol e 5 mM MES. 2. Siliques coleta Aos 7 - ou 8 dias de pós-polinização (DAP), as sementes estão prontas para serem colhidas em meados dos anos para a fase final da embriogênese torpedo e dissecados para endosperma e embrião. Às vezes, pode levar 90-10 DAP se as flores emasculadas são muito jovens. 3. Endosperma e embrião isolando Desde sementes Arabidopsis são pequenos, nós usamos um microscópio de dissecação para isolar endosperma e embrião. Pôr silique 8 DAP-sob um microscópio, use uma pinça para segurar o pedicelo silique e usar a ponta de um outro par de fórceps para deslizar silique a céu aberto na margem onde fundir dois carpelos. Use uma pinça para pegar uma semente em soluções de pH 5,7 a 0,3 M sorbitol e 5 mM MES, faça um pequeno corte no final micrópila a deslizar para fora do embrião, aperte o fim uncut a empurrar para fora endosperma e endosperma separado do tegumento (Kinoshita et al., 2004). Coloque o embrião e endosperma em microtubos separados em nitrogênio líquido. Continue até que acumulamos embrião ou endosperma 10-15 siliques e depois guardar o tubo em um freezer -80 C °. Em alguns casos, endosperma não tem que ser separada da casca da semente, ou seja, pode-se isolar RNA total do endosperma e tegumento mistura para a análise de imprinting gene. III. Seqüenciamento bissulfito 1. Reagentes necessários Brometo de amônio cetiltrimetilamônio (CTAB) para a preparação do DNA genômico, enzimas de restrição, 3 M NaOH (recém-feitos), 6,42 M de uréia / 4 bissulfito de sódio M (2 metabissulfito de sódio M, Sigma-Aldrich, S9000, Na2S2O5, Peso Molecular: 190), 10 mM hidroquinona, um kit de purificação de DNA (Promega Assistente de DNA Sistema Clean-up, Cat. # A7280), TE buffer, 6,3 M NaOH (recém feitos), 10 M NH 4 OAc, 20 mcg / mL tRNA e etanol 100% . 2. Detalhes do protocolo de tratamento bissulfito <ol><li> Endosperma isolar DNA genômico ou embrião através de um procedimento CTAB (Rogers e Bendich, 1988). </li><li> Digest 100 ng - 2 mg de DNA genômico em 20 mL a 100 mL com volume total enzimas de restrição que cortam fora da região a ser analisada. Para o promotor MEA, usamos XhoI, NdeI e PstI ou HindIII. </li><li> Desnaturar as enzimas de restrição do DNA por fervura durante cinco minutos e depois apagar no gelo. </li><li> Adicione 1 / 9 do volume (2,2 mL para 20 mL de DNA digerido) de 3 M NaOH e incubar a 37 ° C por 15 minutos. </li><li> Transferir a solução para um tubo de PCR 250 mL. </li><li> Dissolver 7,5 g de uréia em 10 mL de água destilada estéril; Lentamente, adicionar 7,6 g de metabissulfito de sódio durante 1-2 horas e aquecimento geralmente ajuda a dissolver; Ajustar o pH para 5 com recém feitos 10 M NaOH; Adicionar água destilada estéril para a final volume para 20 mL. Este é 6,24 M de uréia / 4 M de solução de bissulfito de sódio. </li><li> Adicionar 6,24 M de uréia / 4 M de solução de bissulfito de sódio para uma concentração final de 5,36 M e 3,44 M, respectivamente (Paulin et al., 1998). Por exemplo, adicione 208 mL de 6,42 M de uréia / 4 M de solução de bissulfito de sódio para o mL 22,2 acima do DNA genômico desnaturado (Xiao et al., 2003). </li><li> Adicionar 10 mM hidroquinona ao DNA em uma concentração final de 0,5 mM (12 mL para 20 mL a digestão). </li><li> Realizar tratamento em uma máquina de bissulfito PCR: 30 ciclos de 55 ° C por 15 minutos e 95 ° C por 30 segundos. </li><li> Dessalinizar o DNA bissulfito tratado utilizando o Assistente do Sistema DNA Clean-Up da Promega e acompanhar o protocolo (Jacobsen et al., 2000). </li><li> Medir o volume exato de TE recuperado da coluna após dessalinização e adicionar 6,3 M NaOH a uma concentração final de 0,3 M. Incubar a 37 ° C por 15 minutos. </li><li> Adicionar 10 M NH 4 OAc (pH 7,0) para uma concentração final de 3 M, 2 mL de 20 mcg / mL tRNA e 3 volumes de etanol 100% e, em seguida, misture. Centrifugar por 15 minutos a 14.000 rpm. </li><li> Lave o pellet uma vez com etanol 70%, faça uma centrífuga curto, e remover o etanol extra. </li><li> Pellet seco em um speedvac por 5-10 minutos e ressuspender em 25-100 mL tampão TE dependendo de partida quantidade de DNA. O tratado pelo bissulfito de sódio DNA está pronto para a análise PCR. </li></ol> 3. Amplificação por PCR <ol><li> Desde citosinas não metiladas são convertidas em uracila, é difícil para amplificar um fragmento grande usando o DNA tratado pelo bissulfito como um modelo. Assim, costumamos projetar primers para amplificar um produto não superior a 500 pb. Para a seqüência de 4 kb promotor MEA, nós projetamos muitos conjuntos de primers e amplificado 14 fragmentos sobrepostos para cobrir toda a região (Xiao et al., 2003). </li><li> Para seqüenciar o top-fio, na concepção de uma cartilha para a frente, I) escolher um G (guanina) rica região a fim de ter uma maior temperatura de tratamento térmico, sem extras nucleotídeos na primers; II) mudança C (citosina) para Y ( pirimidina) em CG e contextos CNG e alterar o restante para C T (timina). Na concepção de um primer reverso, I), escolha uma região C-ricos; II) para mudar G R (purina) em CG e contextos CNG e alterar o G restantes para A (adenina). </li><li> Para seqüenciar o fundo vertente, na concepção de uma cartilha para a frente, I), escolha uma região C-ricos; II) a mudança para R G em CG e contextos CNG e alterar o G restantes para A. Ao projetar um primer reverso, I) escolha uma região rica em G; II) mudança C para Y (pirimidina) em CG e contextos CNG e alterar o C restantes para T. </li><li> Nós usamos geralmente 1-2 mL do sódio DNA tratado pelo bissulfito como um modelo para cada PCR (Xiao et al., 2003). O produto da PCR deve ser analisado por eletroforese em gel para confirmar o tamanho correto do fragmento, então a ser purificado com gel e clonados no vetor de clonagem TOPO TA pCR2.1 (Invitrogen), como uma inserção. A única colônia é captado e culta; DNA plasmidial extraídos e enviados para o seqüenciamento. </li></ol> 4. Análise da seqüência O princípio do seqüenciamento bissulfito é que citosina unmethylated será convertido para uracil devido à desaminação hidrolítica pela alta concentração de bissulfito de sódio a pH5.0 que será amplificada como timina no produto de PCR, enquanto 5-metil citosina não será modificado por bissulfito de sódio e continuam a ser citosina após a amplificação (Clark et al, 1994;. Frommer et al, 1992).. Depois de obter o resultado de seqüenciamento, que compará-lo com o modelo vertente específica que é usado para a amplificação PCR. Se um resíduo citosina no modelo lê como uma timina no resultado seqüenciamento, isso indica que a citosina não está metilado. Se um resíduo citosina no modelo continua a ser uma citosina no seqüenciamento, isso significa que a citosina é metilado. IV. Resultados representante <img src="/files/ftp_upload/2327/2327fig1.jpg" alt="Figura 1 /" /> Figura 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Flower+Development+in+Arab&#237;dopsis.">Early Flower Development in Arab&#237;dopsis.</a> Plant Cell. 2, 755-767 (1990).</li><li>Xiao, W., Gehring, M., Choi, Y., Margossian, L., Pu, H., Harada, J. J., Goldberg, R. B., Pennell, R. I., Fischer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinting+of+the+MEA+Polycomb+gene+is+controlled+by+antagonism+between+MET1+methyltransferase+and+DME+glycosylase.">Imprinting of the MEA Polycomb gene is controlled by antagonism between MET1 methyltransferase and DME glycosylase.</a> Dev. Cell. 5, 891-901 (2003).</li></ol>

Não há conflitos de interesse declarados.

É relativamente fácil separar embrião do endosperma e tegumento, mas é entediante para separar endosperma de tegumento, especialmente para sementes na fase inicial ou média torpedo da embriogênese. Desde tegumento contribui apenas com uma pequena quantidade de tecido, para alguns genes, por exemplo, MEA e FWA, não temos para separar endosperma de tegumento. Isso significa que podemos isolar RNAs de uma mistura de endosperma e tecidos tegumento, verificar a expressão dos alelos maternos e patern...

Abastecimento <ul><li> Microscópio de dissecação </li><li> Tesoura </li><li> Pinça Ponta Fina </li><li> Tag da jóia </li><li> Estacas de plantas </li><li> Barbante ou fios de arame </li><li> 4 &quot;X 2&quot; X 8 &quot;sacos de polietileno </li><li> 3 &quot;X 1&quot; X 1,0 milímetros Lâminas de microscopia </li><li> Nitrogênio líquido </li><li> Recipientes de azoto líquido </li><li> Bloco de calor </li><li> PCR Tubes </li><li> Termociclador </li><li> Tubos de microcentrífuga </li><li> Microcentrífuga </li><li> Instalação de eletroforese em gel </li><li> Arabidopsis thaliana Columbia-0 Plantas </li><li> Arabidopsis thaliana Landsberg Plantas erecta </li></ul> Reagentes <ul><li> Etanol 70% </li><li> Etanol 95% </li><li> O etanol 100% </li><li> 0,3 M de sorbitol e 5 mM MES-pH 5,7 </li><li> Brometo de amônio cetiltrimetilamônio (CTAB) </li><li> O etanol 100% </li><li> Cholorform </li><li> Enzimas de Restrição </li><li> 3 M NaOH </li><li> 6,3 M NaOH </li><li> 6,24 M de uréia / 4 bissulfito de sódio M </li><li> Destilada estéril H 2 O </li><li> 10 mM hidroquinona </li><li> Assistente de DNA Sistema Clean-Up (Promega) </li><li> 10 M NH 4 OAc </li><li> 20 mcg / mL tRNA </li><li> Tampão TE </li><li> O TOPO TA Cloning Kit (Invitrogen) </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana é um organismo excelente modelo para estudar mecanismos epigenéticos. Uma das razões é o mutante nulo de perda de função de DNA metiltransferases é viável, proporcionando assim um sistema para estudar como a perda da metilação do DNA em um genoma afeta o crescimento e desenvolvimento. Imprinting refere-se a expressão diferencial dos alelos maternos e paternos e desempenha um papel importante no desenvolvimento da reprodução em ambos os mamíferos e plantas. Metilação do DNA é fundamental para determinar se os alelos maternos ou paternos de um gene é expresso impressa ou silenciados. Em plantas com flores, há um evento de fertilização dupla na reprodução: um espermatozóide fertiliza o óvulo para formar um embrião e esperma fusíveis segundo com a célula central para dar origem a endosperma. Endosperma é o tecido onde imprinting ocorre nas plantas. MEDEA, a SET domínio gene grupo Polycomb e FWA, um fator de transcrição que regulam a floração, são os dois primeiros genes mostrado para ser impressa no endosperma e sua expressão é controlada por metilação do DNA e desmetilação em plantas. , A fim de determinar o status de impressão de um gene e padrão de metilação no endosperma, precisamos ser capazes de isolar endosperma primeiro. Desde que a semente é pequena em Arabidopsis, ele continua sendo um desafio para isolar endosperma Arabidopsis e examinar a sua metilação. Neste protocolo de vídeo, relatamos como conduzir um cruzamento genético, para isolar o tecido de endosperma de sementes, e para determinar o estado de metilação de seqüenciamento bissulfito.

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 é um fenômeno em plantas e da reprodução de mamíferos. Metilação do DNA desempenha um papel importante nos mecanismos de imprinting. Isolando endosperma e determinação do estatuto de metilação de genes imprinted em<em&gt; Arabidopsis</em&gt; Pode ser difícil. Neste protocolo, descrevemos como isolar endosperma e determinar a metilação por seqüenciamento bissulfito.

Determinação de metilação de DNA de genes marcados em Arabidopsis Endosperma

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

17.3K visualizações.  Saint Louis University. Imprinting é um fenômeno em plantas e da reprodução de mamíferos. Metilação do DNA desempenha um papel importante nos mecanismos de imprinting. Isolando endosperma e determinação do estatuto de metilação de genes imprinted em Arabidopsis Pode ser difícil. Neste protocolo, descrevemos como isolar endosperma e determinar a metilação por seqüenciamento bissulfito.

Vídeo: 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.

A metilação do DNA: Modificação e Análise de bissulfito

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

Biologia

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: Detecção de alterações numéricas e estruturais em todos os 24 cromossomos humanos ao mesmo tempo usando um ensaio de 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.

A Brasão Cama Ensaio de Sementes para explorar Geneticamente<em&gt; In Vitro</em&gt; Como a germinação Controles do endosperma das sementes em<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.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica 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 ...

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.

A metilação in vitro Ensaio de Proteína Estudo Arginina metilação

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.

Aprimorada Redução Representação Bisulfite Sequencing para a Avaliação de metilação de DNA na resolução de pares de bases

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.

Correlacionando DNA Alterações metilação específico do gene com expressão e transcrição Atividade de astrocítica<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.

Alvejado DNA análise de metilação por Sequenciação de próxima geração

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.

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-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.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

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 ...