The authors have no acknowledgements.

1.標識反応<ol><li> 96ウェルプレートおよび使用の準備-20℃で保存し、製造業者が推奨する濃度において、プレアリコートシアニン3及び5標識dUTPs、非標識ヌクレオチドおよびランダムプライマーを使用する前に。このプロトコルは、各ウェルにdUTPを10.5μlの未標識ヌクレオチドおよびランダムプライマーを標識し、適切のアリコート10.5μlのの目的のために。 </li><li>光から保護し、約1時間、4℃でヌクレオチドおよびプライマーのすぐに使用できる96ウェルプレートを解凍する。 </li><li>解凍後は、光から保護し、少なくとも30分間室温でこのプレートを平衡化する。 </li><li>少なくとも15分間、60℃でDNAサンプルを平衡化する。 </li><li>液体ハンドリングロボットを用いて、96ウェルプレートの各ウェルにヌクレアーゼフリー水を分配する。 注記：水の体積は、各入力DNA試料の濃度に依存し、そして/μlの最終濃度50ngをもたらすのに十分であるべきである。 </li><li>液体漢の使い方dlingロボット、96ウェルプレートのウェルにピペット1μgのDNAは、（1.5を参照）20μlに全量を。 注記：結果データの品質が損なわれる可能性があるが、DNAの小さい量を使用することができる（貴重なサンプルのために、400ngのまでインプットDNA量を使用することができる）。 </li><li>液体ハンドリングロボットを用いて、希釈したDNAのプレートの各ウェルに平衡化ヌクレオチドおよびプライマーの20μlのを転送する。 </li><li> （気密シールは非常に重要です）ストリップキャップで96ウェルプレートをシール。 </li><li>加熱された蓋をPCR機で10分間、99℃でDNAを変性させ、その後アニールするプライマーを氷上で5分間クールスナップ。 </li><li>液体処理ロボットを用いて、徹底的にそれぞれのDNAとピペットミックスにクレノウエキソDNAポリメラーゼ酵素の10μlを添加する。 注：総容積は50μlである必要があります。 </li><li> 96ウェルプレートを密封し、16時間37℃でインキュベートする。 注：4時間の短いインキュベーションしかし十分ですO / Nでcubationはより便利にすることができます。 </li><li>液体ハンドリングロボットを用いて、各ウェルに停止緩衝液の5μlを添加する。 NOTE：ラベリング反応に使用される試薬の概要を表1に示す。 </li></ol>取り込まれなかったヌクレオチドの2の除去<ol><li>シリカメンブレンベースのスピンカラムと専用のスピンカラム処理ロボットを使用して、取り込まれていないヌクレオチドを​​除去（スピンカラムは、手動で処理されてもよい）。製造元の指示に従ってください。 <ol><li> 2ミリリットルチューブに、各ウェルの内容を転送。よくIDをこれらを事前にラベルを付けます。 注：このステップでは、液体ハンドリングロボットを用いて手動で、または好適に行うことができる。 </li><li>スピンカラム処理ロボットを使用している場合は、2ミリリットルチューブ、DNA精製スピンカラムおよび関連するバッファを備えたロボットをロードします。 </li><li>シリカに標識されたDNAを結合するために標識反応を250μlの高塩、DNA結合緩衝液（バッファPB）を追加膜。 </li><li> 500μlの洗浄バッファー（PEをバッファリング）で2回膜を洗浄することにより不純物を除去する。 </li><li> 〜12μLの容量において、精製、標識DNAを回収するために、膜に15μlの低塩の溶出緩衝液を（EBをバッファリング）を追加します。 </li></ol></li></ol> 3. Hybをペアの組み合わせ<ol><li>シアニン3と5標識DNAの適切なペアを組み合わせて、COT-1 DNA、メーカー提供の阻止ミックスと液体処理ロボットを使用して、製造業者が提供するハイブリダイゼーション緩衝液。 注：共有のCNVが問題でない場合、彼らは非常にあるように、これらのHYBペアは、 例えば、私たちは日常的にそのような頭蓋骨癒合症の患者VS腎形成不全患者などの表現型不一致のサンプルを、ペア（サンプルおよび参照、またはサンプル対サンプルであってもよい）同じ臨床的に有意なCNVを運ぶ可能性は低い。参照DNAを使用している場合は、市販のDNA供給が推奨され、それがサンプルと一緒に処理されるべきである。 <ol><li>液体処理ROを使用して、ボット、COT-1 DNA（3333 ngの/μl）を追加し、製造業者が提供する各ウェルに1.1μlのCOT-1 DNA、4.95μlの遮断ミックス24.75を含むように96ウェルプレートの各ウェルに混合し、ハイブリダイゼーションをブロッキング緩衝液μlのハイブリダイゼーション緩衝液。 </li><li>各ウェルに適切なシアニン3標識DNAの9.35を添加する。分注精度を向上させるプリウェット各チップ。 </li><li>各ウェルに適切なシアニン5標識DNAの9.35を添加する。分注精度を向上させるプリウェット各チップ。 </li><li> 1分間、96ウェルプレートボルテックスをシール。各ウェルの底に内容物を収集するために96ウェルプレートをスピン。 </li></ol></li></ol> 4.ハイブリダイゼーション<ol><li> 65℃のハイブリダイゼーションオーブンを予熱し、1バッキングスライドと各アレイスライドの1つのハイブリダイゼーションチャンバーをprewarm。 <ol><li>アレイに適用される前に、プレハイブリダイゼーションインキュベーションで標識されたDNAを変性さ。 24時間、回転オーブンでハイブリダイゼーションを実行します。 </li><li> 30分間、70℃で96ウェルプレートをインキュベートした後、少なくとも5分間37℃で残す。 </li><li> 42℃で加熱されたプラットフォーム上での作業、使用する前に、ハイブリダイゼーションチャンバ内に各バックアップのスライドをロードします。ガスケットスライドの透明部分は、ハイブリダイゼーションチャンバーの「窓」の部分と整列していることを確認してください。 </li><li>ピペットで先に調製したハイブリダイゼーション混合物の42μlをスライドバッキングアレイ上の適切な位置に（セクション3を参照）。分注精度を向上させるプリウェット各チップ。ピペットでゆっくり確認しながら各位置の中心、上に液体がゴムリングの境界に触れていません。 </li><li>バッキングスライド上のすべての位置が満たされると、慎重に上のアレイスライドを下げ、ハイブリダイゼーションチャンバーを組み立てる。それへの書き込みを持つ配列の側はバッキングスライドに直面していることを確認してください。完全にハイブリダイゼーションチャンバーのネジを締めてください。 </li><li>組み立てられたハイブリダイゼーションチャンバーを点検。 Eハイブリダイゼーションチャンバーが垂直方向の端部に載置されたときにゴムリングの境界外のハイブリダイゼーション混合物の漏れ、各気泡がないことをnsureは、高さが約4 mmである。ハイブリダイゼーションチャンバーを回転させた位置で立ち往生し、動いていないに気泡がないことを確認してください。これらのいずれかがある場合は、室のベンチに鋭いタップを与える。すべての気泡が移動していることを確認してください。 </li><li> 24時間、65℃で回転オーブンにハイブリダイゼーションチャンバーを配置します。オーブンロータがバランスしていることを確認します。必要に応じて他の空室を使用しています。 </li></ol></li></ol> 5.洗浄およびスキャンスライド<ol><li>過剰標識DNAを除去した後、各プローブの蛍光を測定するためにスキャンするために、配列を洗ってください。 <ol><li>オーブン逆アセンブルの外にハイブリダイゼーションチャンバーを取る。アレイおよびガスケットスライドが一緒に立ち往生するでしょう。洗浄バッファー1でこれらを沈めると離れて、それらをてこのようにプラスチック、フラットエッジのピンセットを使用しています。 </li><li>ディガスケットスライドをSCARD、バッファ1に沈めラックに配列スライドを配置します。 </li><li>激しく撹拌し、5分間、新鮮な洗浄バッファー1の〜700ミリリットルの配列のスライドを洗う（ノミと磁気攪拌機を使用します）。 </li><li> 〜700ミリリットルの洗浄バッファー2への配列のスライドを移し、激しく撹拌し、90秒間洗浄します。静かに洗浄バッファー2から配列スライドを持ち上げ、彼らはドライ出現する必要があります。 </li><li>スライドプロテクタを使用して、スキャナのスライドホルダーに配列スライドをロードします。アレイスキャナーにスライドをロードし、アレイの製造元の指示に従ってスキャンします。 </li></ol></li></ol>

<ol>
	<li>Miller, D. 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=Consensus+statement:+chromosomal+microarray+is+a+first-tier+clinical+diagnostic+test+for+individuals+with+developmental+disabilities+or+congenital+anomalies.">Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.</a> Am. J. Hum. Genet. 86, 749-764 (2010).</li><li>Ahn, J. W., 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=Array+CGH+as+a+first+line+diagnostic+test+in+place+of+karyotyping+for+postnatal+referrals+-+results+from+four+years'+clinical+application+for+over+8,700+patients.">Array CGH as a first line diagnostic test in place of karyotyping for postnatal referrals - results from four years' clinical application for over 8,700 patients.</a> Mol. Cytogenet. 6 (16), 1755-8166 (2013).</li><li>Ahn, J. W., 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=Validation+and+implementation+of+array+comparative+genomic+hybridisation+as+a+first+line+test+in+place+of+postnatal+karyotyping+for+genome+imbalance.">Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance.</a> Mol. Cytogenet. 3 (9), 1755-8166 (2010).</li><li>Beaudet, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+chromosomal+microarray+analysis+in+developmental+and+behavioral+pediatrics.">The utility of chromosomal microarray analysis in developmental and behavioral pediatrics.</a> Child Dev. 84, 121-132 (2013).</li><li>Park, S. 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=Clinical+implementation+of+whole-genome+array+CGH+as+a+first-tier+test+in+5080+pre+and+postnatal+cases.">Clinical implementation of whole-genome array CGH as a first-tier test in 5080 pre and postnatal cases.</a> Mol. Cytogenet. 4, 12 (2011).</li><li>Iafrate, A. 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=Detection+of+large-scale+variation+in+the+human+genome.">Detection of large-scale variation in the human genome.</a> Nat. Genet. 36, 949-951 (2004).</li><li>Redon, R., 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=Global+variation+in+copy+number+in+the+human+genome.">Global variation in copy number in the human genome.</a> Nature. 444, 444-454 (2006).</li><li>Cafferkey, M., Ahn, J. W., Flinter, F., Ogilvie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+features+in+patients+with+15q11.2(BP1-BP2)+deletion:+Further+delineation+of+an+emerging+syndrome.">Phenotypic features in patients with 15q11.2(BP1-BP2) deletion: Further delineation of an emerging syndrome.</a> Am. J. Med. Genet. A. 164, 1916-1922 (2014).</li><li>Molin, A. M., 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+novel+microdeletion+syndrome+at+3q13.31+characterised+by+developmental+delay,+postnatal+overgrowth,+hypoplastic+male+genitals,+and+characteristic+facial+features.">A novel microdeletion syndrome at 3q13.31 characterised by developmental delay, postnatal overgrowth, hypoplastic male genitals, and characteristic facial features.</a> J. Med. Genet. 49, 104-109 (2012).</li><li>Tropeano, M., 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=Male-biased+autosomal+effect+of+16p13.11+copy+number+variation+in+neurodevelopmental+disorders.">Male-biased autosomal effect of 16p13.11 copy number variation in neurodevelopmental disorders.</a> PLoS One. 8, e61365 (2013).</li><li>Bon, B. W., 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=Further+delineation+of+the+15q13+microdeletion+and+duplication+syndromes:+a+clinical+spectrum+varying+from+non-pathogenic+to+a+severe+outcome.">Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome.</a> J. Med. Genet. 46, 511-523 (2009).</li></ol>

The authors have nothing to disclose.

アレイCGHは、三倍体としてバランスのとれた再構成または倍数性異常を検出しません。さらに、低レベルのモザイク不均衡が検出されないことがある。しかし、アレイCGHは、多くの細胞遺伝学研究室で置き換えたG-バンド染色体分析よりCNVを検出するためのより高い解像度を有する。したがって、ゲノムワイドなCNV検出のための現在のゴールドスタンダードです。これは、将来的には、次世?...

これは、欠失または染色体セグメントの余分なコピーが知的障害、異形症とcongentical奇形を引き起こすことを長年にわたって知られており、いくつかのケースでは、遺伝的症候群1を引き起こしてきた。しかし、これらの変化のゲノムワイド検出のため2000年代半ばまで、利用可能な唯一の技術は、不均衡の地域や性質に応じて、5-10Mb周りの解像度を持つG-バンド染色体分析、で、そのことはできません材料が同じバンドパターンと異なる染色体から地域によって置き換えられている異常な染色体を検出する。そのようなin situハイブリダイゼーションと多重ライゲーション特異的プローブ増幅における蛍光として補助的な細胞遺伝学的技術が疑われる特定の症候不均衡例では、特定の遺伝子座の尋問のために利用されてきたが、それは、アレイ比較ゲノムハイブリダイゼーション（アレイCGH）の導入までではなかった日常の臨床診断の中にコピー数変異体（のCNV）のゲノムワイド検出が（通常は120キロバイト程度）を大幅に増加した解像度で可能になったervice 2-5。一部の地域では正常な集団6-7で普及しているための調査研究と並んで臨床サービス作業は、他のCNVながら、以前に検出できない、そのような自閉症やてんかん8-11などneurodisabilitiesと関連している、のCNVことが示されている。 この論文に記載されているプロトコルは、当社の英国国民保健サービス（NHS）の臨床診断検査室で使用されている。我々はこの状態が資金提供サービスでコストを最小限に抑える新たなハイブリダイゼーション戦略、バッチテストやロボットを使用しています。 以下に詳述するプロトコールに先立って、高品質のDNAを、適当な出発物質、一般的に、血液、培養細胞又は組織サンプルから抽出されるべきである。 280吸光度比（bにすべき：分光光度計は濃度を測定するために使用することができ、260をチェックし（&gt; 50 ngの/μlのあるべき）E 1.8から2.0）。ゲル電気泳動は、DNAが有意な劣化なしに高分子量であることを確認するために使用することができる。 このプロトコルは、自動液体処理ロボットを使用して実行ごとに96個のサンプルを標識しているより高いスループット実験室用に設計されています。しかし、自動化することなく、低いスループット·ラボに適合させることができる。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CGH microarray 8 x 60K</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">G4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH wash buffers</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH hybridisation buffer</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Minelute purification kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">28006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH labelling kit</td>
			<td style="width:71px;">Enzo</td>
			<td style="width:119px;">ENZ-42672-0000</td>
			<td></td>
		</tr>
	</tbody>
</table>

array comparative genomic hybridization (array cgh) for detection of genomic copy number variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

ハイブリダイズしたアレイ上の各プローブは、赤と緑の蛍光色素の混合物として可視化される（ 図1参照）。各プローブのための緑色蛍光信号に赤の比率は、スキャナによっ​​て定量化され、関連するソフトウェアは、それらのゲノム位置に応じてLOG2比としてこれらをプロットし、プリセットの境界の外に落下する領域を特定します。結果の配列トレースは、ゲノムのアンバ?...

Watch this Scientific Journal Video about Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants at JoVE.com

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

ゲノムコピー数変異の検出のためのアレイCGHは、Gバンド核型分析に取って代わった。本論文では、技術や診断サービスの研究室への応用について説明します。

ゲノムコピー数変異体の検出のためのアレイ比較ゲノムハイブリダイゼーション（アレイCGH）

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

array-comparative-genomic-hybridization-array-cgh-for-detection

Research

JoVE Journal

Biology

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

19.4K ビュー.  Guy's & St Thomas' NHS Foundation Trust. ゲノムコピー数変異の検出のためのアレイCGHは、Gバンド核型分析に取って代わった。本論文では、技術や診断サービスの研究室への応用について説明します。 

ビデオ: Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Isolation of Genomic DNA from Mouse Tails

マウスの尾からのゲノムDNAの単離

生物学

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全ゲノムアレイ比較ゲノムハイブリダイゼーションの技術デモンストレーション

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

慢性のためのマイクロドライブアレイ in vivoで録音：ドラ​​イブの作製

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大規模な挿入環境ゲノムライブラリーの製造

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNAマイクロアレイ：サンプルの品質管理、アレイハイブリダイゼーションとスキャニング

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

ゲノムMRI - ゲノムDNA内の配列パターンを研究するためのパブリックリソース

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

バーコード酵母ライブラリーの競争力のあるゲノム画面

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

SPLINTERを使用してプールされたシーケンスからレアゲノム変異の検出

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryoteのゲノム形質転換<em&gt; Ostreococcusタウリ</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

ゲノムDNAから5  - ヒドロキシの選択的捕獲

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...