Name: optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing
Uploaded: 2017-07-28T12:30:00
Description: Watch this Scientific Journal Video about Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing at JoVE.com

우리는 미국 농무부 농업 연구청과 국립 과학 재단 (IOS 1025881 및 IOS 1361554)으로부터 자금을 지원하고 싶습니다. 우리는 R. Caspers에게 온실 유지 관리 및 식물 관리에 감사드립니다. 우리는 Illumina 라이브러리 준비 및 시퀀싱이 수행 된 Minnesota Genomics Center 대학에도 감사드립니다. 저널 편집자와 4 명의 익명의 평론가가 우리 원고를 더욱 강화시킨 것에 대해 감사드립니다. 우리는 또한 OECD가 일본의 동료들과 협력 프로젝트를 위해 이러한 프로토콜을 통합하는 SK와의 협력에 감사한다.

1. 세포질 분리 및 DNA 추출을위한 식물 재료의 생성 <ol><li> 밀 묘목의 표준 성장 <ol><li> 구석 당 4 ~ 6 개 종자가있는 작고 정방형의 냄비에 질석에 식물 종자를 심습니다. 온실 또는 성장 챔버로 16 시간주기, 23 ℃ / 18ºC 밤으로 옮긴다. </li><li> 매일 식물에 물을주십시오. 발아시 및 발아 후 7 일째에 ½ 티스푼의 과립 20-20-20 NPK 비료로 식물을 비옥하게하십시오. </li></ol></li><li> 밀 ?...

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지금까지 대부분의 organellar sequencing 연구는 특정 DC를 풍부하게하는 전통적인 DC 방법에 초점을두고 있습니다. 모스 ( 40)를 포함하여 다양한 식물로부터 세포 소기관을 분리하는 방법이 설명되었다. 밀 15 및 귀리 11 과 같은 단핵구; 아라비돕시스 11 , 해바라기 17 , 유채 14 등의 족자입니다. ?...

<html> 게놈 시퀀싱은 중요한 식물 형질의 기본 유전 적 기초를 분석하는 강력한 도구입니다. 대부분의 게놈 시퀀싱 연구는 대부분의 유전자가 핵에 위치하므로 핵 게놈 내용에 초점을 맞 춥니 다. 그러나, (진핵 생물에서) 미토콘드리아와 색소체 포함 organellar 게놈 (식물, 전문화 된 형태로, 엽록체, 광합성에서 작동)는 생명체의 개발, 스트레스 반응 및 전반적인 체력 1에 필수적인 중요한 유전 정보를 기여한다. Organellar 게놈은 일반적으로 핵 게놈 시퀀싱을위한 총 DNA 추출에 포함되지만 DNA 추출 이전에 세포 소기관 수를 줄이는 방법도 사용됩니다 2 . 많은 연구에서 전체 gDNA 추출물의 sequencing 결과를 사용하여 organellar 게놈 3 , 4 , 5 ,xref &quot;&gt; 6 , 7. 그러나 연구 대상이 세포 소기관 게놈에 초점을 맞추고있을 때 많은 gDNA가 핵산 염기 서열에&quot;손실 &quot;되어 있기 때문에 전체 핵산을 사용하면 시퀀싱 비용이 증가합니다. 적당한 게놈, bioinformatically 8 도전 2에 관한 것이다. 또한, 염기 서열의 정확한지도 위치를 해결 중복 핵 게놈 내로 소기관 사이 organellar 서열의 전사에 의한 읽기. 핵 게놈 organellar 게놈의 정제 하나이다 전략을 사용하여 미토콘드리아와 엽록체 사이의 상 동성 영역으로 매핑되는 판독을 분리 할 수 ​​있습니다. 많은 식물 종의 세포 소기관 게놈이 서열화되어 있지만, 소기관 게놈 다양성의 폭은 거의 알려져 있지 않습니다야생 개체군 또는 경작 된 육종 풀에서 이용 가능하다. Organellar 게놈은 또한 반복 서열 9 사이의 재조합으로 인해 중요한 구조적 재 배열을 거친 동적 분자로 알려져있다. 또한, 세포 소기관 게놈의 여러 사본이 각 세포 소기관에 포함되어 있으며, 여러 세포 소기관이 각 세포 내에 포함되어 있습니다. 이 게놈의 모든 사본이 동일하다는 것은 이질균이라고 알려져 있습니다. &quot;마스터 서클&quot;의 정경과는 달리 하위 게놈 서클, 선형 염색체, 선형 concatamers 및 가지 형 구조 10을 포함하여 organellar 게놈 구조의 더 복잡한 그림에 대한 증거가 증가하고 있습니다. 식물 세포 소기관 게놈의 조립은 상대적으로 큰 크기와 상당한 거꾸로 된 직접 반복에 의해 더욱 복잡해진다. 세포 기관 분리, DNA 정제 및 후속 유전체에 대한 기존의 프로토콜<html> 전자 시퀀싱은 종종 성가 시며 많은 양의 조직을 필요로하며 수백 g의 어린 잎 조직을 출발점 11 , 12 , 13 , 14 , 15 , 16 , 17로 필요로 합니다. 이렇게하면 조직이 제한되어있을 때 세포 내 게놈 시퀀싱을 사용할 수 없게됩니다. 어떤 상황에서는, 세대 간 (through generational basis) 또는 교차점을 통해 유지되어야하는 수컷 멸균 계통에서 필요할 때와 같이, 종자의 양은 제한적이다. 이러한 상황에서 세포 내 소기관 DNA를 정제 한 다음 전체 게놈 증폭을 수행 할 수 있습니다. 그러나 전체 게놈 증폭은 구조적 변이, 하위 게놈 구조 및 이종성 수준을 평가할 때 특히 중요한 문제인 시퀀싱 바이어스를 도입 할 수 있습니다&gt; 18. 짧은 읽기 시퀀싱 기술에 대한 라이브러리 준비의 최근 발전은 전체 게놈 증폭을 피하기 위해 낮은 입력 장벽을 극복했습니다. 예를 들어, Illumina Nextera XT 라이브러리 준비 키트는 1 ng의 적은 DNA를 입력으로 사용할 수 있습니다 19 . 그러나 PacBio 또는 Oxford Nanopore 시퀀싱 기술과 같은 장시간 시퀀싱 응용 프로그램을위한 표준 라이브러리 준비에는 여전히 많은 양의 입력 DNA가 필요하며 이는 세포 내 게놈 시퀀싱에 대한 도전이 될 수 있습니다. 최근에, 입력량을 줄이고 마이크로 그램 - 양의 DNA를 얻기가 어려운 시료에서 게놈 시퀀싱을 용이하게하기 위해 사용자가 작성한 긴 판독 시퀀싱 프로토콜이 개발되었습니다 20 , 21 . 그러나, 고 분자량의 순수한 세포 내 소 분획을 이들 라이브러리 제제에 공급하는 것은 여전히 ​​어려운 과제이다. 우리는o 전체 게놈 증폭없이 NGS에 적합한 세포 내 DNA 농축 및 분리 방법을 비교 및 ​​최적화합니다. 구체적으로, 우리의 목표는 잎의 하위 샘플과 같이 제한된 출발 물질로부터 고 분자량 세포 기관 DNA를 풍부하게하는 우수 사례를 결정하는 것이 었습니다. 이 연구는 organellar DNA를 풍부하게하는 방법의 비교 분석을 제시한다 : (1) 상업적으로 이용 가능한 DNA CpG- 메틸 결합 도메인 단백질 풀다운 접근의 사용에 기초한 DNA 분획 프로토콜 (2) 수정 된 전통적인 분별 원심 분리 프로토콜 22 식물 조직에 적용 23 . 우리는 다른 식물 및 조직 유형으로 쉽게 확장 될 수있는 밀엽 조직으로부터 세포 기관 DNA를 분리하는 최상의 방법을 권장합니다.

<table border="0" cellpadding="0" cellspacing="0" width="685">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2-mercaptoethanol (beta-mercaptoethanol; BME)</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:119px;">M3148-100ml</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2-propanol (Isopropyl alcohol/isopropanol), bioreagent</td>
			<td>Sigma Aldrich</td>
			<td>I9516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">agarose, Bio-Rad Cetified Megabase agarose</td>
			<td>Bio-Rad</td>
			<td>1613108</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">analytical balance</td>
			<td>Mettler Toledo</td>
			<td>AB54-S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">balance</td>
			<td>Mettler Toledo</td>
			<td>PB1502-S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>B4287-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ceramic grinding cylinders, 3/8in x 7/8in</td>
			<td>SPEX SamplePrep</td>
			<td>2183</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cryogenic Blocks compatible with tissue homogenizer for holding 50 ml tubes</td>
			<td>SPEX SamplePrep</td>
			<td>2664</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNaseI</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ethanol, absolute</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylenediamine Tetraacetic Acid (EDTA), 0.5M Solution, pH8.0</td>
			<td>Fisher</td>
			<td>BP2482-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gel imaging system</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">gel stain</td>
			<td></td>
			<td></td>
			<td style="width:167px;">Such as GelRed or Ethidium Bromide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">grinding pestle, wide tip for 2 ml conical tubes</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Guanidine-HCl, 8M solution</td>
			<td>ThermoFisher</td>
			<td>24115</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LightCycler 480 SYBR Green I Master</td>
			<td>Roche</td>
			<td>4707516001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">liquid nitrogen</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing enzymes from Trichoderma harzianum</td>
			<td>Sigma</td>
			<td>L1412</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td>G Bioscience</td>
			<td>24115</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">magnetic rack</td>
			<td>ThermoFisher</td>
			<td>A13346</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">microcentrifuge tubes, LoBind 1.5 ml&#160;</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">microcentrifuge tubes, standard nuclease-free 1.5 ml</td>
			<td>Eppendorf</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:216px;">microcentrifuge, refrigerated</td>
			<td>Sorvall&#160;</td>
			<td>Legend X1R</td>
			<td style="width:167px;">Or equivalent product, must be capable of reaching at least 18,000 x g with rotors for 50 ml tubes, Oak Ridge tubes, and 1.5 ml tubes</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:216px;">microcentrifuge, room temperature</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td style="width:167px;">Or equivalent product, must be capable of reaching at least 18,000 x g with rotor for 1.5 ml and 2 ml microcentrifuge tubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microcon DNA Fast Flow Centrifugal Filter Units</td>
			<td>EMD Millipore</td>
			<td>MRCFOR100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Miracloth, 1 square per sample cut to fit funnel</td>
			<td>EMD Millipore</td>
			<td>475855</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NEBNext Microbiome DNA Enrichment Kit</td>
			<td>New England Biolabs</td>
			<td>E2612L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">parafilm</td>
			<td>Parafilm M</td>
			<td>PM992</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic pots and trays</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polyvinylpyrrolidone (PVP)</td>
			<td>Fisher</td>
			<td>BP431-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td>Qiagen</td>
			<td>19131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pulsed-Field Gel Electrophoresis rig (e.g. CHEF DR III)</td>
			<td>Bio-Rad</td>
			<td>1703697</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">purification beads, Agencourt AMpureXP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAamp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51304</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen 20/g Genomic Tip DNA Extraction Kit</td>
			<td>Qiagen</td>
			<td>10223</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qiagen Buffer EB (elution buffer)</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qiagen DNA Extraction Buffer Set</td>
			<td>Qiagen</td>
			<td>19060</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QiaRack</td>
			<td>Qiagen</td>
			<td>19015</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">qPCR machine (e.g. Roche Light Cycler 480)</td>
			<td>Roche</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qPCR plate sealing film</td>
			<td>Roche</td>
			<td>4729757001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qPCR plate, 96 well plate</td>
			<td>Roche</td>
			<td>4729692001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit&#160;assay tubes</td>
			<td>Life Technologies</td>
			<td>Q32856</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit Broad Spectrum assay kit</td>
			<td>Life Technologies</td>
			<td>Q32850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Qubit High Sensitivity assay kit</td>
			<td>Life Technologies</td>
			<td>Q32851</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNaseA</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Serological pipettes (20 ml) and pipet-aid</td>
			<td>Fisher</td>
			<td>13-678-11</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small funnels, 1 per sample</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Chloride</td>
			<td>Ambion</td>
			<td>AM9759</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Soft paintbrush, 2 per sample</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="260">
			<td height="260" style="height:260px;width:216px;">SPEX SamplePrep 2010 Geno/Grinder or another type of tissue homogenizer</td>
			<td>SPEX SamplePrep</td>
			<td></td>
			<td style="width:167px;">Or another comparable tissue homogenizer. If you do not have access to a tissue homogenizer, then grinding in a pre-chilled mortar and pestle will suffice (see protocol for details). However, a homogenizer will give more consistent results and total homogenization time is reduced.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td>Omnipure</td>
			<td>8550</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TBE</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">thermomixer</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td>Sigma</td>
			<td>T2819-100ml</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tube rotater</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tubes, 50 mL conical polypropylene</td>
			<td>Corning</td>
			<td>352070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tubes, 50 ml high-speed polypropylene&#160;</td>
			<td>ThermoScientific/Nalgene</td>
			<td>3119-0050</td>
			<td style="width:167px;">e.g. Nalgene Oakridge tubes or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">vermiculite</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water bath</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">water, sterile and certified Nuclease-free&#160;</td>
			<td>Fisher</td>
			<td>1481</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water, sterile milliQ</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

optimization and comparative analysis of plant organellar dna enrichment methods suitable for next-generation sequencing

식물 세포 소기관 게놈은 복잡한 구조 및 / 또는 서브 게놈 조각을 형성하기 위해 페어링 또는 재조합을 거칠 수있는 크고 반복적 인 요소를 포함합니다. Organellar 게놈은 또한 주어진 세포 또는 조직 유형 (heteroplasmy) 내의 혼합물에 존재하며, 아형의 다량은 발육 기간 동안 또는 스트레스하에있을 때 (부분 화학량 론적 이동) 변화 할 수있다. 세포 기관 게놈 구조와 기능에 대한 더 깊은 이해를 위해서는 차세대 시퀀싱 (NGS) 기술이 필요합니다. 전통적인 sequencing 연구는 organellar DNA를 얻기 위해 몇 가지 방법을 사용합니다 : (1) 대량의 시작 조직이 사용되면 균질화되고 차등 원심 분리 및 / 또는 기울기 정화가 이루어집니다. (종자, 재료 또는 공간이 한정되어있는 경우, 즉) (2) 조직의 작은 양이 사용된다면, 동일한 프로세스는 (1) 충분한 DNA를 얻기 위해 전체 게놈 증폭 다음과 같이 수행된다. (3) 생물 정보학 분석은 seq전체 게놈 DNA를 분석하고 세포 기관 판독을 분석합니다. 이러한 모든 방법에는 고유 한 문제점과 단점이 있습니다. (1)에서 이와 같이 많은 양의 시작 조직을 얻는 것이 어려울 수 있습니다. (2)에서, 전체 게놈 증폭은 시퀀싱 바이어스를 도입 할 수있다; (3)에서 핵과 세포 내 게놈 간의 상동 성은 집합과 분석을 방해 할 수있다. 거대한 핵 게놈을 가진 식물에서, organellar DNA를 풍부하게하여 생물 정보학 분석을위한 서열화 비용 및 서열 복잡성을 줄이는 것이 유리하다. 여기에서 전통적인 차동 원심 분리 방법과 네 번째 방법 인 CpG- 메틸 풀다운 방식을 비교하여 총 게놈 DNA를 핵 및 세포 내 분획으로 분리합니다. 두 가지 방법 모두 미토콘드리아와 엽록체의 비율이 다르긴하지만 세포 내 소기관 서열에 대해 고도로 농축 된 DNA 인 NGS에 충분한 양의 DNA를 생산합니다. 우리는 밀 잎 조직을위한 이러한 방법의 최적화를 제시하고 주요 이점과 d샘플 입력, 프로토콜 용이성 및 다운 스트림 응용 프로그램의 맥락에서 각 접근법의 단점.

이 원고에 제시된 프로토콜은 식물 조직에서 세포 기관 DNA를 풍부하게하는 두 가지 방법을 설명합니다. 여기에 제시된 조건은 밀의 조직 최적화를 반영합니다. 프로토콜의 주요 단계, 필요한 조직 입력 및 DNA 출력의 비교는 그림 1에 설명되어 있습니다. 우리가 테스트 한 DC 프로토콜의 단계는 이전에 설명한 것과 비슷한 조건을 따른다 ( <strong class="x...

Watch this Scientific Journal Video about Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing at JoVE.com

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

2 가지 plant organellar DNA 농축 방법의 비교 및 ​​최적화가 제시됩니다 : 전통적인 차동 원심 분리 및 메틸화 상태에 기초한 총 gDNA 분획. 우리는 결과로 나오는 DNA 양과 품질을 평가하고, 짧은 읽기 차세대 시퀀싱에서의 성능을 시연하며, 장시간 읽기 단일 분자 시퀀싱에서의 사용 가능성에 대해 논의합니다.

차세대 시퀀싱에 적합한 식물성 세포질 DNA 농축 방법의 최적화 및 비교 분석

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

The overall goal of this experiment is to compare two methods used to isolate high quality organellar DNA, both plastid and mitochondria DNA from plant leaf tissue that is suitable for next-generation sequencing. These methods can help answer key questions in plant biology such as the extent of organellar diversity across taxa and how changes in organellar genome sequence or structure affect development and stress response. We will compare two techniques, the differential centrifugation method that enriches for one specific organellar type and the meta fractionation technique that recovers total organellar DNA from smaller frozen tissue samples.The standard procedure for growing wheat seedlings, is to plant seeds in vermiculite, in square pots with four to six seeds per corner. Transfer the pots to a greenhouse with a 16 hour light cycle. Water the plants each day.Fertilize the plants with one quarter teaspoon of granular 20-20-20 MPK fertilizer upon germination, and again at seven days post germination. For adulation of wheat seedlings, plant seeds and care for plants as in the standard procedure. But place the pots in a dark growth chamber.An alternative to growing these seedlings at a dark growth chamber is to cover the plants in the greenhouse but with proper ventilation. The first step in this procedure is to isolate the organelles. Harvest five grams of fresh tissue and rinse it in cold, sterile water in a chilled beaker on ice.Using scissors, cut the leaf tissue into approximately one centimeter pieces directly into a pre-chilled 50 millimeter tube containing two ceramic grinding cylinders. Keep the samples on ice throughout the procedure. To avoid cross-contamination, it is important to change the scissors between samples.Take the samples to the fume hood and add 20 milliliters of STE buffer to each 50 milliliter tube. Place the samples into pre-chilled cryogenic grinding blocks in a tissue grinding apparatus and grind the samples for 30 seconds at 1, 750 rotations per minute. Place the samples on ice for about one minute.Rotate the sample positions and grind for another 30 seconds at 1, 750 rotations per minute. Return the samples to the ice bucket. For each sample, insert a funnel into a clean 50 milliliter tube placed in ice and place one layer of filtration cloth into the funnel.Pre-wet the filtration cloth with five milliliters of STE buffer and save the flow through. Pour the homogenized tissue into the funnel. Rinse the grinding tube with 15 milliliters of STE buffer, recap and invert the tube to rinse walls and lid and pour its contents into the funnel.Carefully remove the ceramic stones and then squeeze and wring out the filtration cloth into the funnel. To avoid cross-contamination, change gloves between samples. Wrap the tube caps with paraffin film to avoid spillage.Centrifuge at 2, 000 x G, at four degree celsius for 10 minutes. Next, use a serological pipette to carefully aspirate the supernatant without disturbing the pellet and place it in a 50 milliliter high speed centrifuge tube with a tight sealing gasket. Place a small beaker of ice on a scale, tear the scale and weigh each supernatant.Use STE buffer to balance the tubes to within 0.1 grams and then centrifuge at 18, 000 x G in four degree celsius for 20 minutes. When the centrifugation is complete, return samples to the fume hood and discard the supernatant. Add one milliliter of ST buffer to the pellet and resuspend gently using a soft paintbrush.Add 24 milliliters of ST buffer to get a final volume of 25 milliliters. Gently swirl the paintbrush around in a suspension to mix, and then press the paintbrush on the side of a tube to remove all liquid before removing the paintbrush. After balancing the tubes, centrifuge at 18, 000 x G in four degree celsius for 20 minutes.During this centrifugation, prepare DNA's 1 solution as described in the text protocol and aliquot 200 microliters to a 1.5 milliliter tube per sample. When the centrifugation is complete, discard the supernatant and blot the tube. To each high speed centrifuge tube, add 300 microliters of STE buffer and resuspend the pellet using a soft paint brush.Place the paint brush in the previously prepared 1.5 milliliter tube containing 200 microliters of DNA's 1 solution and swirl the paint brush to remove any residual pellet stuck in the brush. Pipette the DNA's 1 solution into the high speed centrifuge tube and gently swirl to mix. Wrap paraffin film around the top of each tube and incubate at 37 degrees celsius in a water bath for 30 minutes.During the incubation, gently mix by swirling. Using a pipette tip with a wide orifice, gently pipette the pellet mixture out of the tube and place it in a 1.5 milliliter low binding tube. Add 500 microliters of 400 millimolar EDTA, pH 8.0 to the high speed centrifuge tube.Gently pipette to completely remove the residual pellet and transfer the EDTA to the low binding tube containing the pellet mixture. Gently mix by inversion. Centrifuge at 18, 000 x G, at four degree celsius for 20 minutes.Subsequently, discard the supernatant, blot each tube and use the pellet immediately for DNA isolation. In this method, organellar DNA is enriched from total genomic DNA that was extracted using commercial DNA extraction columns. Prepare the required amount of MBD2 FC protein bound magnetic beads, per the manufacturer's instructions and scale the reactions to use between one and two micrograms of total input DNA.In this demonstration, 160 microliters of beads is required for one microgram of total input DNA. To capture methylated nuclear DNA, add one microgram of input DNA to a tube containing 160 microliters of MBD2 bound magnetic beads for each individual sample and gently pipette up and down with a wide bore tip. Add the appropriate volume of 5x bind wash buffer for a final concentration of 1x and pipette the sample up and down with a wide bore tip, a few times to mix.Rotate the tubes at room temperature for 15 minutes. For the success of the pull down, it is critical to thoroughly mix the beads during the incubation, particularly, if there is significant bead clumping. To prevent bead clumping and to ensure pull down of the methylated DNA, gently pipette the samples with a wide bore pipette tip and flick the samples two to three times throughout the incubation.Briefly spin the tubes containing the DNA and magnetic bead mixture. Place the tubes on a magnetic rack for at least five minutes to collect the beads to the side of the tubes. The solution should appear clear, it contains un-methylated organellar enriched DNA.Using wide bore tips, carefully remove the cleared supernatant without disturbing the beads and transfer the supernatant to a clean, low binding two milliliter microcentrifuge tube. Store the samples at 20 or 80 degrees celsius. Acute PCR assay was employed to assess the relative abundances of three organelle specific implicons in total genomic DNA and the organellar DNA fraction obtained from both methods.Overall, the data indicate that the differential centrifugation method, preferentially enriches for mitochondria. The unmethylated fraction of the methylfractionated total genomic DNA, shows substantial enrichment of both organellar implicons. Percentages of reads mapped to the mitochondria or chloroplast chinese spring and chris cultivated wheat reference genomes are consistent with the QPCR results.Differential centrifugation yield DNA that is more enriched in mitochondria DNA and methylfractionation yields DNA that likely reflects the native abundance of the two organellar genomes. Sample labels including E, designate idyllated samples and NE designate non-idyllated samples. Interestingly, idyllation did not change organellar abundances.In both methods, the theoretical coverage of the mitochondria or chloroplast referenced genomes, exceeds 100x coverage, even when 12 libraries atremultiplexed. The total genomic DNA migrates as a diffused smear in post field gel electrophoresis and exceeds 50 kilobases. The nuclear fraction after methylfractionation decreases in size, but remains centered around 50 kilobases, suggesting that methylfractioanted DNA is most suitable for long read sequencing.Once mastered, both organellar DNA extraction techniques can be done in one to two days, from sample collection to assessment of final product, if performed properly. The methylfractionation technique has the potential to greatly expand the range of organellar DNA studies to rare and difficult samples, since tissue inputs are extremely low. However, differential centrifugation may still be preferable when solely mitochondria DNA is required for downstream studies.Likewise, centrifugation steps maybe adjusted to preferentially extract plastid DNA if desired. Both organellar DNA extraction methods yield DNA that is suitable for next generation sequencing but methylfractionation yields high molecular rate DNA that is ideal for long read sequencing.

Research

JoVE Journal

Genetics

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One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

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

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Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

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Rare Event Detection Using Error-corrected DNA and RNA Sequencing

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

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차세대 시퀀싱의 원형 염색체 구조를 사용 하 여 유전자 규제 시퀀스의 높은 처리량 Id (4 C-seq)를 캡처

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

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

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Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...