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.

곰팡이 재료 1. 준비 <ol><li> 흰가루병 성장 <ol><li> 다음의 조건으로 성장 챔버에 식물을 성장 : 22 ° C 일 온도 20 ℃ 밤 온도, 80 % 상대 습도, 14 시간 낮 길이 125 μE / m 2 / s의 광 강도 (형광등에 의해 제공된 ). </li><li> 18, 29 설명 Golovinomyces cichoracearum 변형 UCSC1 오이 식물 (쿠쿠 미스 사티의 다양한 부시 챔피언) 감염. </li></ol></li><li> 수확...

<ol>
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가지 의무적 biotrophic 흰가루병 균 순수한 고 분자량의 게놈 DNA를 얻기 위해, 전술 한 방법의 변형 된 버전 (30)를 개발 하였다. 이 최적의 프로토콜을 이용하여 평균 수율은 150 mg의 분생 당 7 μg의 DNA를, 종래의 프로토콜로 얻어진 수율의 두배이다. 또한, 평균 크기는 48.5 KBP에 걸쳐 약 20 KBP에서 증가했다. 이 프로토콜은 cucumber- G.의 cichoracearum 시스템에 최적화되었다. biotrophic...

<html> 흰가루병은 가지 의무적 biotrophic 곰팡이 식물의 그룹이 함께 할 때, 식물 병 전 세계적으로 하나의 가장 큰 원인, 즉 병원균이다. 분류 학적으로 가족 Erisyphaceae 2에서 다섯 개 종족으로 분류 된 흰가루병의 900 설명 종있다. 경제적 중요성과 그들의 호스트와 개발 친밀한 관계를 모두 인해, 흰가루병 질환&gt; 100 년 연구의 대상이되어왔다. 감염시, 흰가루병이 병원균 혜택을, 세포의 구조, 신진 대사와 그 호스트의 분자 생물학의 급격한 변화를 유도. 그러나, 흰가루병의 연구는 그들의 가지 의무적 라이프 스타일, 그리고 순수 문화에 곰팡이의 성장은 아직 5, 4, 3에 설명되지 않은에 특히 도전,&quot;외부 참조&quot;&gt; 6, 7, 8. 과도 변환이 어떤 종 9, 10에보고되었지만 흰가루병의 신뢰성, 안정적인 유전자 변형은 아직 완성되지 않았습니다. 흰가루병 게놈의 염기 서열 및 조립으로 인해 게놈 자체의 기능들이 어려운 입증했다. 다른 진균 유전체로 - (180 MBP 120) 기준으로 60 구성 - 흰가루병 게놈이 크고 90 % 고르게 분포 반복 소자 (11). 이러한 요소는 비 긴 말단 반복뿐만 아니라 분류 중복 요소를 포함한다. 하나의 흰가루병 종, Blumeria 그라 미니 f를 두 formae SPECIALES. SP. hordei 및 F. SP. 소맥 (년 Bgh 각각 BGT)뿐만 아니라 포도 흰가루병에 에리시 페 necator, 꿀벌이N 서열, 및 다른 여러위한 초안 게놈은 12, 13, 14을 완성 하였다. 게놈의 반복적 인 특성은 조립 어렵게 있으며, 완성 된 게놈 년 Bgh 2의 L50 메가 12 6,989 supercontigs로 조립 하였다. 큰 게놈에도 불구하고, 흰가루병은 각각 년 Bgh 및 BGT 예측 5845 개 및 6540 유전자, 단백질 코딩 유전자의 수가 적은 것으로 보인다. 시퀀싱 흰가루병은 생존 11, 12, 13, 14 그들의 숙주 식물에 진균의 의존성과 일치 다른 진균류에 필수적인 최소한 99 핵심 유전자 결여 보인다. 텔로미어, 중심절 리보솜 RN 근처 반복 서열유전자 배열과 전이 될 풍부한 영역이 제대로 짧은 판독 시퀀싱 전략 조립 및 언더 표현 게놈 어셈블리 (15)에 종종있다. 이러한 영역은 게놈 서열에서 발생하는 공극의 많은 책임을 생각하고, 이것이 반복 요소 (16)의 그 광범위한 팽창으로 흰가루병에 적용된다. 높은 플라스틱 게놈 영역은 종종 반복적 인 지역 3에서 발견된다. 이들은 염색체 재 배열 사이트로서 기능하고 종종 이펙터 단백질을 코딩하는 유전자 및 이차 대사 효소를 코딩하는 유전자와 같은 강력한 선택 압력하에 유전자를 인코딩한다. 단일 분자 긴 읽기 시퀀싱 기술의 발전은 유전체 (15)의 반복적 인 지역에 걸쳐 순서에 대한 잠재적 인 솔루션을 제공합니다. 예를 들어, Faino 등. (2015)을 포함하여 긴 시퀀스 기술과 광 m 읽어 발견apping 유전자 주석 유전자 주석의 수를 증가 (및 일부의 수를 감소 시키거나 누락 게놈의 반복 DNA 서열의 비율 배로 그들을 곰팡이 병원균 Verticillium 달리아 두 균주 갭리스 게놈 시퀀스를 생성하도록 허용 ) 및 계시 게놈 17 재구성. 최소 크기&gt; 20 KBP 이러한 긴 읽기 시퀀싱 기술, 고품질의 게놈 DNA의 높은 농도를 사용하려면 필요하다. 여기에서 우리는 분생 포자로부터 포자 수집, 고 분자량의 DNA의 정제를위한 우리의 방법을 설명하고 흰가루병 종 Golovinomyces cichoracearum를 사용하여 우리의 품질 관리 평가는 오이 (18)에 성장. 이 프로토콜은 켈러 B. 실험 그룹 (쮜리히 대학, 스위스 취리히) (13), (19)에서 개발 된 프로토콜에 기초증가 DNA 수율 주도 여러 변형 및 크기의 DNA&gt; 48.5 KBP의 높은 비율을 포함한다. 이 프로토콜은 또한 연구소 20, 21, 22 에너지 공동 게놈의 미국학과의 추천에 따라 품질 관리 단계를 포함한다. 용해 완충액에 포함 된 각 시약 및 각 정제 단계에 근거하는 기능 등 (2008) 23 헨리에 기재되어있다. 식물과 미생물 조직에서 고 분자량 게놈 DNA의 분리에 사용할 수 게시 된 프로토콜은 또한이 프로토콜 24, 25, 26, 27, 28의 설계시 협의했다.

<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

분체 곰팡이 곰팡이는 경제적으로 중요한 곰팡이 식물 병원균의 그룹입니다. 상대적으로 작은 인해 잘 발달 된 유전자와 게놈 자원의 부족 부분에 분자 생물학 및 병원체의 유전에 대한 알려져있다. 이 생물의 게놈 시퀀싱 및 조립 엄청나게 어려운 만든 대형, 반복적 인 게놈을 가지고있다. 여기서는 하나의 흰가루병 종 Golovinomyces cichoracearum에서 고 분자량의 게놈 DNA의 수집, 추출, 정제 및 품질 관리에 대한 평가 방법을 설명한다. 기술 된 프로토콜은 최적화 된 페놀 / 클로로포름 게놈 DNA를 추출하여 포자의 기계적 파괴를 포함한다. 전형적인 수율은 150 mg을 분생 당 7 μg의 DNA에 있었다. 이 절차를 이용하여 분리 된 게놈 DNA는 긴 판독 순서 (즉,&gt; 48.5 KBP)에 적합하다. 품질 관리 대책이 게놈 DNA의 크기, 수율 및 순도를 보장하기 위해 또한이 방법에 기재된. 여기에 설명 된 품질의 게놈 DNA의 염기 서열은 조립 및 차례로 더 나은 이해로 이어질 것입니다 여러 흰가루병 유전체의 비교를이 농업 병원균의 제어를 개선합니다.

G.로부터 정제 된 게놈 DNA를 60ng의 대표적인 예는 겔 전기 영동을 사용하여 아가로 오스 겔상에서 실행 cichoracearum 및 펄스 필드 겔 전기 영동을 사용하는 각각도 1 및도 2에 나타낸다. 소폭 통과하고 품질 관리를 실패 패스 게놈 DNA 제제를 나타낸다. 완벽한 품질 관리를 통과 게놈 DNA의 준비는 긴 읽기 게놈 시퀀싱 방법을 ?...

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.

긴 읽기 시퀀싱에 대한 흰가루병에서 높은 분자량의 게놈의 DNA의 정제

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.

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Genetics

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환자 조직에 대한 메틸 결합 DNA 캡처 시퀀싱

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