Name: purification of high molecular weight genomic dna from powdery mildew for long-read sequencing
Uploaded: 2017-03-31T13:00:00
Description: Watch this Scientific Journal Video about Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing at JoVE.com

This protocol was developed in support of a Joint Genome Institute-Community Sequencing Project (#1657). The work was supported in part by the Philomathia Foundation, the National Science Foundation (#0929226) and the Energy Biosciences Institute to S. Somerville; and by Swiss National Science Foundation (#310030_163260) to B. Keller. We would like to thank our colleagues, M. Figureroa and M. Miller (University of Minnesota), R. Panstruga (RWTH Aachen University), C. Pedersen (University of Copenhagen), P. Spanu (Imperial College), J. Taneja (University of California Berkeley), M. Wildermuth (University of California Berkeley), R. Wise (Iowa State University) and S. Xiao (University of Maryland) for their generous advice and for volunteering their protocols as we developed the protocol described here.

1.真菌材料的制备<ol><li>成长白粉病<ol><li>生长在生长室的植物在以下条件下：22℃白天温度，20℃夜间温度，80％相对湿度，14-H昼长和125μE/米2 / s的光强度（由荧光灯提供）。 </li><li>感染的黄瓜植物（ 黄瓜苜蓿为Bush Champion）与高氏cichoracearum应变UCSC1如所描述的18，29。 </li></ol></li><li>收获白粉病分生孢子<ol><li>从感染的植物收获的...

<ol>
	<li>Agrios, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology. , (1969).</li><li>Braun, U., Cook, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Taxonomic manual of the Erysiphales (Powdery Mildews). , (2012).</li><li>Both, M., Csukai, M., Stumpf, M. P. H., Spanu, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+Blumeria+graminis+indicate+dynamic+changes+to+primary+metabolism+during+development+of+an+obligate+biotrophic+pathogen.">Gene expression profiles of Blumeria graminis indicate dynamic changes to primary metabolism during development of an obligate biotrophic pathogen.</a> Plant Cell. 17 (7), 2107-2122 (2005).</li><li>Glawe, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+powdery+mildews:+A+review+of+the+world's+most+familiar+(yet+poorly+known)+plant+pathogens.">The powdery mildews: A review of the world's most familiar (yet poorly known) plant pathogens.</a> Annu Rev Phytopathol. 46, 27-51 (2008).</li><li>Fabro, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+profiling+Arabidopsis+at+the+stage+of+Golovinomyces+cichoracearum+haustorium+formation.">Genome-wide expression profiling Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation.</a> Plant Physiol. 146 (3), 1421-1439 (2008).</li><li>H&#252;ckelhoven, R., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+the+plant-powdery+mildew+interaction.">Cell biology of the plant-powdery mildew interaction.</a> Curr Opin Plant Biol. 14 (6), 738-746 (2011).</li><li>Micali, C. O., Neumann, U., Grunewald, D., Panstruga, R., O'Connell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+a+specialized+plant-fungal+interface+during+host+cell+internalization+of+Golovinomyces+orontii+haustoria.">Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria.</a> Cell Microbiol. 13 (2), 210-226 (2011).</li><li>Chandran, D., Inada, N., Hather, G., Kleindt, C. K., Wildermuth, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+Arabidopsis+cells+at+the+powdery+mildew+infection+site+reveals+site-specific+processes+and+regulators.">Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators.</a> Proc Natl Acad Sci. 107 (1), 460-465 (2010).</li><li>Spanu, P. D., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Powdery+mildew+genomes+in+the+crosshairs.">Powdery mildew genomes in the crosshairs.</a> New Phytol. 195 (1), 20-22 (2012).</li><li>Vela-Corc&#237;a, D., Romero, D., Tor&#233;s, J. A., De Vicente, A., P&#233;rez-Garc&#237;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=Transient+transformation+of+Podosphaera+xanthii+by+electroporation+of+conidia.">Transient transformation of Podosphaera xanthii by electroporation of conidia.</a> BMC Microbiol. 15 (20), (2015).</li><li>Hacquard, S., Francis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Botanical Research. 70, 109-142 (2014).</li><li>Spanu, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+expansion+and+gene+loss+in+powdery+mildew+fungi+reveal+tradeoffs+in+extreme+parasitism.">Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.</a> Science. 330 (6010), 1543-1546 (2010).</li><li>Wicker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wheat+powdery+mildew+genome+shows+the+unique+evolution+of+an+obligate+biotroph.">The wheat powdery mildew genome shows the unique evolution of an obligate biotroph.</a> Nat Genet. 45 (9), 1092 (2013).</li><li>Jones, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+genomic+structural+variation+in+the+grape+powdery+mildew+pathogen,+Erysiphe+necator.">Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator.</a> BMC Genomics. 15, 1081 (2014).</li><li>Thomma, B. P. H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind+the+gap;+seven+reasons+to+close+fragmented+genome+assemblies.">Mind the gap; seven reasons to close fragmented genome assemblies.</a> Fungal Genet Biol. 90, 24-30 (2016).</li><li>Parlange, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+invasion+of+transposable+elements+accounts+for+the+large+size+of+the+Blumeria+graminis+f.sp.+tritici+genome.">A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome.</a> Funct Integr Genomics. 11, 671-677 (2011).</li><li>Faino, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+real-time+sequencing+combined+with+optical+mapping+yields+completely+finished+fungal+genome.">Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome.</a> mBio. 6, (2015).</li><li>Adam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Erysiphe+cichoracearum+and+E.+cruciferarum+and+a+survey+of+360+Arabidopsis+thaliana+accessions+for+resistance+to+these+two+powdery+mildew+pathogens.">Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens.</a> Mol Plant Microbe In. 12 (12), 1031-1043 (1999).</li><li>Bourras, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+avirulence+loci+and+allele-specific+effector+recognition+control+the+PM3+race-specific+resistance+of+wheat+to+powdery+mildew.">Multiple avirulence loci and allele-specific effector recognition control the PM3 race-specific resistance of wheat to powdery mildew.</a> Plant Cell. 27 (10), 2991-3012 (2015).</li><li>Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant genotyping II: SNP technology. , (2008).</li><li>Healey, A., Furtado, A., Cooper, T., Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+a+simple+method+for+extracting+next-generation+sequencing+quality+genomic+DNA+from+recalcitrant+plant+species.">Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.</a> Plant Methods. 10 (21), (2014).</li><li>Fang, G., Hammar, S., Grumet, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+inexpensive+method+for+removing+polysaccharides+from+plant+genomic+DNA.">A quick and inexpensive method for removing polysaccharides from plant genomic DNA.</a> Biotechniques. 13 (1), 52-54 (1992).</li><li>Jordon-Thaden, I. E., Chanderbali, A. S., Gitzendanner, M. A., Soltis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+CTAB+and+TRIzol+protocols+improve+RNA+extraction+from+chemically+complex+Embryophyta.">Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.</a> Appl Plant Sci. 3 (5), 1400105 (2015).</li><li>Murray, M. G., Thompson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+high+molecular+weight+plant+DNA.">Rapid isolation of high molecular weight plant DNA.</a> Nucleic Acids Res. 8 (19), 4321-4325 (1980).</li><li>Greco, M., S&#225;ez, C. A., Brown, M. T., Bitonti, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+method+for+high+quality+co-extraction+of+genomic+DNA+and+total+RNA+from+low+biomass+Ectocarpus+siliculosus,+the+model+brown+alga.">A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.</a> PLoS One. 9 (5), e96470 (2014).</li><li>Wilson, I. W., Schiff, C. L., Hughes, D. E., Somerville, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+analysis+of+powdery+mildew+disease+resistance+in+the+Arabidopsis+thaliana+accession+Kashmir-1.">Quantitative trait loci analysis of powdery mildew disease resistance in the Arabidopsis thaliana accession Kashmir-1.</a> Genetics. 158 (3), 1301-1309 (2001).</li></ol>

为了从专性活体营养性白粉病菌获得纯的，高分子量的基因组DNA中，先前所描述的方法的修改版本被开发30。使用该优化的协议的平均产量是每150毫克分生孢子7微克DNA，与现有协议中得到的产率加倍。另外，平均尺寸从大约20 kbp的增加到超过48.5 kbp的。该协议是在黄瓜- G. cichoracearum系统进行了优化。为了获得在其他白粉病最好的质量和纯度最高的DNA或专性活体营养性真菌?...

<html>白粉病是一组专性活体营养真菌植物致病菌，当合起来，为植物病害全球1的最大原因。有白粉病的超过900种描述，已经在分类学上分为家庭Erisyphaceae 2中的五个部落。两个原因，一是经济上的重要性，他们用自己的主机发展的亲密关系，白粉病病一直为&gt;百年研究的课题。感染后，白粉菌引起的细胞结构，代谢及宿主分子生物学剧变，受益这种病原体。然而，白粉病的研究是特别具有挑战性的，因为它们专生活方式，并且在纯培养的真菌生长的尚未描述3，4，5，“外部参照”&gt; 6，7，8。白粉病的可靠，稳定的遗传转化还尚未完成，虽然瞬时转化已经在一些物种中9，10的报道。 白粉病基因组测序和组装已经证明，很难因数量的基因组本身的特性。白粉病基因组是大的（120 - 180 MBP）相对于其它真菌的基因组，和包括60 - 90％的均匀分布的重复元件11。这些元件包括非长末端重复，以及未分类的重复元件。单白粉病品种， 白粉病的F两个专化型 。 SP。 大麦和f。 SP。 小麦（BGH和白粉菌，分别地）以及葡萄白粉病葡萄白粉菌，有蜂ñ测序，以及其他几个人的基因组草案已经完成12，13，14。基因组的重复性质取得了组装困难，并且已完成的基因组BGH组装到6989个supercontigs具有2 MB 12的L50。 尽管有大量的基因组中，白粉病似乎具有少量的蛋白质编码基因，分别在BGH和白粉菌预测，5845倍6540的基因。测序白粉病似乎也缺少了在其他真菌，这是对他们的寄主植物的真菌生存11，12，13，14依赖性一致至关重要至少99核心基因。 近端粒，着丝粒，核糖体RN重复序列A基因阵列和在可转座元件富集区域是从短读测序策略组装不良和低于限额在基因组组件15常常。这样的区域被认为是负责许多发生在基因组序列的间隙，并且这适用于白粉病与它们广泛重复元件16的膨胀。高塑性的基因组区域在这种重复区域3经常发现。它们充当的染色体重排的一个位点和通常编码在强选择压力的基因，如编码效应蛋白的基因和编码次生代谢的酶的基因。在单分子长读测序技术的进步提供了跨越基因组15的重复区域测序潜在的解决方案。例如，Faino 等。 （2015）发现，其中包括长序列读技术和光学米apping允许他们产生的间隙少的基因组序列为植物病原真菌黄萎病大丽花的两个菌株，三倍基因组中的重复DNA序列的比例，增加基因注释的数量（和减少的部分的数目或丢失的基因注解）和揭示基因组重排17。 为了采用这些长读测序技术，高浓度的高品质的基因组DNA，以最小的尺寸&gt; 20 kbp的，是必要的。在这里，我们描述了我们的分生孢子收集，高分子量DNA的纯化方法从分生孢子和我们使用白粉病品种高氏cichoracearum质量控制评估生长在黄瓜18。该协议是基于B·凯勒实验室集团（苏黎世大学，苏黎世瑞士）13，19开发的协议和包括几个修饰导致增加的DNA产量和DNA&gt; 48.5千碱基对大小的比例较高。该协议还包括基于来自能源联合基因组的美国国防部研究所20，21，22项建议的质量控制措施。 包括在裂解缓冲液中的各试剂，并且对于每个纯化步骤的基本原理的功能，在亨利（2008）23所描述的。用于从植物和微生物组织高分子量的基因组DNA的分离可用公开的方案本协议24，25，26，27，28的设计期间进行了协商。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MM400 Ball Mill</td>
			<td>Retsch</td>
			<td>20.745.0001</td>
			<td>Or equivalent ball mill</td>
		</tr>
		<tr>
			<td>2mL tubes for ball milling</td>
			<td>Sarstedt&#160;</td>
			<td>72.694.007</td>
			<td></td>
		</tr>
		<tr>
			<td>5/32&#34; stainless steel milling balls</td>
			<td>OPS Diagnostics</td>
			<td>GBSS 165-5000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprocessor controlled 280 Series Water Bath Preset to 65&#176;C&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>2825</td>
			<td>Or equivalent water bath</td>
		</tr>
		<tr>
			<td>Microprocessor controlled 280 Series Water Bath Preset to 37&#176;C</td>
			<td>Thermo Fisher Scientific</td>
			<td>2825</td>
			<td>Or equivalent water bath</td>
		</tr>
		<tr>
			<td>Eppendorf 5417R refrigerated microcentrifuge</td>
			<td>Krakeler Scientific&#160;</td>
			<td>38-022621807</td>
			<td>Or equivalent microcentrifuge</td>
		</tr>
		<tr>
			<td>Chlorform:IAA (24:1 v/v)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C0549</td>
			<td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:IAA (25:24:1 v/v)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15593031</td>
			<td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td>
		</tr>
		<tr>
			<td>100% Isopropanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34923</td>
			<td>Chill to -20&#176;C prior use</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase, Dnase-free (10mg/ml)</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium metabisulfite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Lauryl Sacroinate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>L9150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Cetyltrimethyl ammonium bromide (CTAB)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H6269</td>
			<td></td>
		</tr>
		<tr>
			<td>Uvex by Honeywell Futura Goggles S345C, Uvextreme</td>
			<td>Staples</td>
			<td>423263</td>
			<td>Or equivalent eyewear</td>
		</tr>
		<tr>
			<td>High Five COBALT nitrile gloves, LARGE</td>
			<td>Neta Scientific</td>
			<td>HFG-N193</td>
			<td>Or equivalent gloves</td>
		</tr>
		<tr>
			<td>GelRed Nucleic Acid Gel Stain 10,000X in water</td>
			<td>Biotium</td>
			<td>41003</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide 10mg/ml</td>
			<td>Biotium</td>
			<td>40042</td>
			<td>Hazardous in case of skin contact. Wear nitrile gloves and protective eyewear</td>
		</tr>
		<tr>
			<td>&#160;Agarose Ultrapure Bioreagent</td>
			<td>VWR International LLC</td>
			<td>JT4063</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1kb Plus DNA Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERSM1332</td>
			<td></td>
		</tr>
		<tr>
			<td>8-48kb CHEF DNA size standards</td>
			<td>Bio-Rad</td>
			<td>170-3707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippin Prep&#160;&#160;</td>
			<td>Life Technologies Corporation</td>
			<td>4472172</td>
			<td>Or equivalent pulse-field gel aparatus</td>
		</tr>
		<tr>
			<td>Whatman&#160;qualitative filter paper, Grade 1</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>WHA1001042</td>
			<td>Or equivalent growth chamber</td>
		</tr>
		<tr>
			<td>Percival&#160; Growth Chamber</td>
			<td>Percival</td>
			<td>AR66LXC9</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 8000 UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-8000-GL</td>
			<td>Or equivalent spectrophotometer</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>P11496</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE Buffer (Tris-acetate-EDTA) (50X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>B49</td>
			<td>Dilute 1/50 with water before use (to 1X)</td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>12090015&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Borate-EDTA, 10X Solution (Electrophoresis)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP13334</td>
			<td>Dilute 1/10 with water before use (to 1X)</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td>Liquid nitrogen (-196 &#176;C) is a freezing hazard. It will also expand rapidly upon warming and should not keep in a tightly closed container</td>
		</tr>
	</tbody>
</table>

purification of high molecular weight genomic dna from powdery mildew for long-read sequencing

白粉病菌是一类重要经济价值的植物病原真菌。相对知之甚少的分子生物学和这些病原体的遗传学，部分原因是由于缺乏发达的遗传和基因组资源。这些有机体具有大的，重复的基因组，这取得了基因组测序和装配极其困难。在这里，我们描述了从一个白粉病物种， 高氏cichoracearum高分子量的基因组DNA的集合，提取，纯化和质量控制评估方法。所描述的协议包括孢子，随后优化的苯酚/氯仿基因组DNA提取的机械破坏。典型的产率是每150个毫克分生孢子7微克DNA。正在使用此过程中分离的基因组DNA是适合长时间读测序（ 即 ，&gt; 48.5 kbp的）。质量控制措施，以确保尺寸，收量，以及基因组DNA的纯度也在该方法中描述的。这里所描述的质量的基因组DNA的测序将允许组件和多个白粉病的基因组，这反过来将导致更好地理解的比较和改进了这一农业病原体的控制。

60ng从G.纯化的基因组DNA的代表性实例cichoracearum使用凝胶电泳在琼脂糖凝胶上运行，并用脉冲场凝胶电泳在图1和2，分别被示出。基因组DNA制备物通，勉强通过和失败的质量控制的代表。基因组DNA制备物，充分通过质量控制是理想的使用长读基因组测序方法测序。制剂，该制剂通过轻微的质量控制是可接受的，但制剂，充...

Watch this Scientific Journal Video about Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing at JoVE.com

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

The overall goal of this protocol is to isolate high molecular weight genomic DNA from powdery mildew haploid conidia for long-read genome sequencing. This method can answer key questions in the plant pathogenic fungal biology field, such as the structure and composition of hard to study genomes. The main advantage of this technique is that it allows for the extraction of high-quality genomic DNA from a pathogen that is difficult to work with.To begin the experiment, quickly remove steel balls from conidia cryovials with a magnet. Add 700 microliters of 65 degree Celsius lysis buffer and vortex the solution for five to 10 seconds. After five to 10 seconds, a slurry is formed.Add 300 microliters of 65 degree pre-warmed 5%volume by volume sarkosyl and gently invert it five times. Incubate the tubes for 30 minutes at 65 degrees Celsius. Using a wide bore pipette tip, transfer the entire solution to a new two milliliter microcentrifuge tube.Add one volume of chloroform isoamyl alcohol to the lysis solution, and gently invert the tube to mix five times. Incubate the mixture for 10 minutes at room temperature. Gently invert the tube to mix five times at the halfway point, and again at the end of the incubation.Next, centrifuge and room temperature for 15 minutes. Using a wide bore pipette tip, carefully transfer the aqueous layer to a new two milliliter microcentrifuge tube, and avoid including material from the interface. Add 750 microliter volume of room temperature 100%isopropanol and gently invert the tube six times.Then centrifuge the mixture at room temperature for 15 minutes. Carefully remove the supernatant and discard it. Add 450 microliters pre-chilled 70%ethanol.Centrifuge the mixture for five minutes at room temperature. Carefully remove the supernatant and discard it. Centrifuge at 1000 times g for three seconds.Carefully remove the supernatant with a fine pipette tip and discard the supernatant. Air dry the pellet for 15 minutes at room temperature. Resuspend the pellet in 300 microliters of TE.Incubate the DNA overnight at four degrees Celsius.The next morning, gently flick to resuspend any DNA that has not gone into solution. To remove RNA contamination, add 10 microliters RNase. Gently invert the tube three times and centrifuge the DNA at 1000 times g for three seconds.Then incubate the DNA at 37 degrees Celsius for two hours. Add 300 microliters phenol chloroform isoamyl alcohol and gently invert to mix five times and incubate the DNA for 10 minutes at room temperature. Gently invert the tube to mix the DNA five times at the halfway mark and again at the end of the incubation.Next centrifuge the tube for 15 minutes at room temperature. Using a wide bore pipette tip transfer the supernatant to a new 1.5 milliliter microcentrifuge tube. Add three microliters of 3 molar sodium acetate pH 5.2 then gently invert the tube five times.Add 750 microliters of pre-chilled 100%ethanol and gently invert the tube five times. Incubate the mixture overnight at 20 degrees Celsius. The following morning, centrifuge the tube for 30 minutes at four degrees Celsius.Carefully remove and discard the supernatant. Add 450 microliters of pre-chilled 70%ethanol. Then centrifuge the tube for five minutes at four degrees Celsius.After pelleting the DNA, carefully remove the supernatant. Spin the tube again at 1000 times g for three seconds to bring down residual supernatant. Remove residual supernatant with fine tipped pipette.Air dry the pellet for 30 to 60 minutes at room temperature. After air drying, resuspend the pellet in 27.5 microliters of TE.The next day, gently flip to resuspend any residual material that did not go into solution. Aliquot 2.5 microliters into 22.5 microliters TE for quality control tests.Store the DNA samples at four degrees Celsius until submission for sequencing. For long term storage, store samples at 80 degrees Celsius and minimize freeze thaw cycles to prevent shearing. Using the protocol, purified genomic DNA was used to run gel electrophoresis which obtained DNA that passed quality control with a strong single band above 20kbp and minimal smearing, DNA that gave a marginal result with a single band running above 20kbp, and a failed result that displays smearing below 20kbp and includes fragments at 100bp.Pulsed-field gel electrophoresis, or PFGE, was performed to distinguish between an acceptable DNA sample with a majority of DNA greater than 48.5kbp and a marginal DNA sample with the majority of DNA between 20 and 48.5kbp. Following this procedure, other methods like single molecule long-read sequencing can be performed to answer additional questions about the powdery mildew genome.

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...