この研究は、米国国立衛生研究所（CHとNS051630/MH076090/MH078972にPJにGM071440）によって部分的にサポートされていました。

1。ゲノムDNAの断片化ゲノムワイドなシーケンシングプラットフォームに適した所望のサイズ範囲に超音波処理を用いてゲノムDNA断片。 （私達は通常〜300 bpまで超音波処理。）を1％アガロースゲル（ 図1）上の断片化したゲノムDNAのサイズ分布を確認します。 2。 DNAの調製ゲノムDNA中の5-HMCの豊富に基づく出発DNAの量を決定します。 5-HMCのレベルが異なる組織型においても大きく変動するので、出発DNA量は、試料の5-HMCのレベルによって異なります。例については、 表1を参照してください。 3。 β-GTの触媒反応（グルコース転移反応） <ol><li> 1時間37℃の水浴中で表2の詳細かつインキュベートした混合物をピペッティング。 </li><li>インキュベーション後、当たり10μgのDNAを用いて、QIAクイックヌクレオチド除去キットを使用した反応をクリーンアップコラム。列あたり30μlの水で溶出し、組み合わせる。 </li></ol> 4。ビオチン化反応（化学は、ここをクリックします） <ol><li> 150μMの（ すなわち 、30μlのDNA溶液当たりの作業溶液5μl）の最終濃度に溶出したDNA溶液中のDBCO-SS-PEG3 -ビオチンコンジュゲートワーキング溶液（1mM）を（ステップ3から）を追加します。 </li><li> 2時間37℃の水浴中でピペッティングとインキュベートしてミックス。 </li><li> QIAクイックヌクレオチド除去キットとの反応をクリーンアップします。理想的な総溶出量は100μlです。 </li><li>マイクロスケール分光光度計（ 例えば 。、光度計）を用いて回収したDNA量を定量化する。 </li></ol> 5。 5-HMC-含むDNAの捕捉<ol><li>製造元の指示に従って、1XのB＆W緩衝液1mlでのDynabeadsストレプトMyOne C1の50μlを3回洗浄する。マグネチックスタンドでビーズを分離。 </li><li>回収されたビオチン化Dに2X B＆Wの等量のバッファーを追加洗浄したビーズに、NA（100 UL）。 </li><li>ローテーターで穏やかに回転しながら室温で15分間インキュベートする。 </li><li>マグネチックスタンドでビーズを分離し、1X B＆W緩衝液1mlでビーズを3回洗浄する。 </li><li>ローテーターで穏やかに回転しながら室温で2時間、新たに調製し、50mMのDTTを100μlのビーズをインキュベートすることによってDNAを溶出する。 </li><li>マグネチックスタンドでビーズを分離。 DTTを除去するために、製造指示に従ってマイクロバイオスピンカラムに溶離液6と負荷を吸引します。標的DNAは現在のソリューションである。 </li><li>のEB緩衝液10μl中にキアゲンMinElute PCR精製キット、溶出DNAによる前工程から溶出したDNAを精製する。濃度が20 ngの/ ULより高ければ量子ビット蛍光光度、または光度計を用いてDNAを定量化する。 DNAは下流のゲノムワイドなシーケンシングライブラリの準備のための準備ができています。 </li></ol> 6。代表的な結果品質O IFfのゲノムDNAが高く、β-GTとビオチン化反応後の典型的な回収率は約60から70パーセントである。しかし、捕獲効率は試料の5-HMCのレベルに応じてさまざまな種類の組織と大きく異なります。一般的に、脳のゲノムDNAの捕捉効率は約4から9パーセントであり、いくつかの極端な場合には、効率が12％にまで達する可能性があります。 ES細胞については、平均捕集効率は、神経幹細胞のための〜0.5％とは対照的に、〜2-4％である。これまで見て最も低い効率は、がん細胞からのゲノムDNAのためだった。すべての濃縮されたDNAは、標準の次世代ライブラリ調製のプロトコルのための準備ができています。関連のプライマーが使用可能な場合だけでなく、捕獲されたDNAはまた、インプットDNAと比較して、いくつかの断片の濃縮を検出するリアルタイムPCR用テンプレートとして使用することができます。 <img alt="図1" src="/files/ftp_upload/4441/4441fig1.jpg" /> 図1。中で超音波処理したヒトゲノムDNA断片1％アガロースゲル。1X TE緩衝液120μlのヒトiPS細胞から単離したゲノムDNA10μgを超音波処理装置（Covaris）を使用して超音波処理した。超音波処理後、超音波処理したDNAの2μlを超音波処理したDNA断片の大きさを比較するためのDNAマーカーの100 bpを使用して1％アガロースゲルにロードした。 <table border="1"><tbody><tr><td> コンポーネント </td><td> ボリューム </td><td> 最終濃度 </td></tr><tr><td>水</td><td> _μL </td><td></td></tr><tr><td> 10 Xのβ-GTの反応バッファー</td><td> 2μlの</td><td> 1つのX </td></tr><tr><td>最大10μgのゲノムDNAへ</td><td> _μL </td><td>最大500 ng /μlに</td></tr><tr><td> UDP-6-N 3 - GLC（3mM）を</td><td> 0.67μlの</td><td> 100μMの</td></tr><tr><td> β-GT（40μM） </td><td> 1μlの</td><td> 2μM </td></tr><tr><td>全体積</td><td> 20μlの</TD&gt; <td></td></tr></tbody></table>組織のゲノムDNA（高5-HMCコンテンツ&gt; 0.1％）のi） <table border="1"><tbody><tr><td> コンポーネント </td><td> ボリューム </td><td> 最終濃度 </td></tr><tr><td>水</td><td> _μL </td><td></td></tr><tr><td> 10 Xのβ-GTの反応バッファー</td><td> 10μlの</td><td> 1つのX </td></tr><tr><td>最大20μgのゲノムDNAへ</td><td> _μL </td><td>最大500 ng /μlに</td></tr><tr><td> UDP-6-N3-GLC（3mM）を</td><td> μlの1.33 </td><td> 100μMの</td></tr><tr><td> β-GT（40μM） </td><td> 2μlの</td><td> 2μM </td></tr><tr><td>全体積</td><td> 40μlの</td><td></td></tr></tbody></table> ⅱ）幹細胞のゲノムDNA（中央値5-HMCコンテンツ〜0.05％）の場合<table border="1"><tbody><tr><td>コンポーネント</td><td>ボリューム</td><td>最終濃度</td></tr><tr><td>水</td><td> _μL </td><td></td></tr><tr><td> 10 Xのβ-GTの反応バッファー</td><td> 10μlの</td><td> 1つのX </td></tr><tr><td>最大50μgのゲノムDNAへ</td><td> _μL </td><td>最大500 ng /μlに</td></tr><tr><td> UDP-6-N3-GLC（3mM）を</td><td> 3.33μlの</td><td> 100μMの</td></tr><tr><td> β-GT（40μM） </td><td> 5μlの</td><td> 2μM </td></tr><tr><td>全体積</td><td> 100μlの</td><td></td></tr></tbody></table> iii）癌細胞のゲノムDNA（低5-HMCコンテンツ〜0.01％）の場合 表1：インプットDNA選択的化学標識法による種々の5-HMCレベルで試料を用いてラベリング反応の量の例。 <table border="1"><tbody><tr><td> サンプル </td><td> 5-HMCレベル </strong&gt; </td><td> 出発DNA（μg）を </td><td> 標識後リカバリ（ビーズへの入力）（μg）を </td><td> 回収率 </td><td> プルダウンDNA（NG） </td><td> プルダウン得 </td></tr><tr><td>成体マウス小脳</td><td> 0.4パーセント</td><td> 10 </td><td> 7.5 </td><td> 75パーセント</td><td> 236 </td><td> 3.1パーセント</td></tr><tr><td>生後7日目のマウスの小脳</td><td> 0.1パーセント</td><td> 11 </td><td> 9 </td><td> 82パーセント</td><td> 140 </td><td> 1.6パーセント</td></tr><tr><td>マウスES細胞はE14 </td><td> 0.05パーセント</td><td> 60 </td><td> 42 </td><td> 70パーセント</td><td> 350 </td><td> 0.8パーセント</td></tr></tbody></table> 表2。マウス脳組織およびES細胞からの代表的な結果。

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5 - ヒドロキシメチル（5 - HMC）は、特定の哺乳類の細胞型における実質的な量で最近同定されたエピジェネティックな修飾の存在しています。ここで紹介する方法は、5 - HMCのゲノムワイドな分布を決定するためのものです。我々は5-HMCのヒドロキシル基にアジド基を含有する人工グルコース部分を転送するために、β-グルコシルT4バクテリオファージを使用しています。アジド基を化学的...

<table border="1"><tbody><tr><td> 名前 </td><td> 会社 </td><td> カタログ＃ </td><td> コメント </td></tr><tr><td></td><td></td><td></td><td> 試薬 </td></tr><tr><td> 5M塩化ナトリウム（NaCl） </td><td>プロメガ</td><td> V4221 </td><td></td></tr><tr><td> 0.5M pH8.0エチレンジアミン四酢酸（EDTA） </td><td>プロメガ</td><td> V4231 </td><td></td></tr><tr><td> 1Mトリズマベース（トリス）pH7.5 </td><td>インビトロジェン</td><td> 15567-027） </td><td></td></tr><tr><td> HEPES 1M、pH7.4の</td><td>インビトロジェン</td><td> 15630 </td><td></td></tr><tr><td> 1M塩化マグネシウム（MgCl 2） </td><td>アンビオン</td><td> AM9530G </td><td></td></tr><tr><td>ジメチルスルホキシド（DMSO） </td><td>シグマ</td><td> D8418 </td><td></td></tr><tr><td>ツイーン20 </td><td>フィッシャーBioReagents </td><td> BP337-100 </td><td></td></tr><tr><td> DBCO-SS-PEG3 - ビオチンコンジュゲート</td><td>化学ツール]をクリックし</td><td> A112P3 </td><td></td></tr><tr><td> 1,4 - ジチオスレイトール、超純（DTT）の超純粋な</td><td>インビトロジェン</td><td> 15508-013 </td><td></td></tr><tr><td> QIAクイックヌクレオチド除去キット</td><td>キアゲン</td><td> 28304 </td><td></td></tr><tr><td>マイクロバイオスピンカラム6 </td><td>バイオ·ラッド</td><td> 732-6222 </td><td></td></tr><tr><td>ダイナMyOne </td><td>インビトロジェン</td><td> 650から01 </td><td></td></tr><tr><td>ストレプトアビジンC1 </td><td></td><td></td><td></td></tr><tr><td>キアゲンMinElute PCR精製キット</td><td>キアゲン</td><td> 28004 </td><td></td></tr><tr><td>超高純度アガロース</td><td>インビトロジェン</td><td> 16500500 </td><td></td></tr><tr><td> UDP-6-N 3-グルコース</td><td>アクティブモティ</td><td> 55013 </td><td></td></tr><tr><td></td><td></td><td></td><td> 酵素 </td></tr><tr><td> （β-GT）は、β-グルコ</td><td>ニューイングランドBiolab </td><td> M0357 </td><td></td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td>超音波処理装置</td><td> Covaris </td><td></td><td></td></tr><tr><td>デスクトップの遠心分離機</td><td></td><td></td><td></td></tr><tr><td>水浴</td><td>フィッシャー·サイエンティフィック</td><td></td><td></td></tr><tr><td>ゲルランニング装置</td><td>バイオ·ラッド</td><td></td><td></td></tr><tr><td> NanoDrop1000 </td><td>サーモサイエンティフィック</td><td></td><td></td></tr><tr><td> Labquakeチューブシェーカー</td><td> Barnstead </td><td></td><td></td></tr><tr><td> Labquakeチューブシェーカー</td><td> Thermolyne </td><td></td><td></td></tr><tr><td>磁気分離スタンド</td><td>プロメガ</td><td> Z5342 </td><td></td></tr><tr><td>量子ビット2.0蛍光計</td><td>インビトロジェン</td><td></td&gt; <td></td></tr><tr><td></td><td></td><td></td><td> 試薬のセットアップ 10 Xのβ-GTの反応緩衝液（500mMのHEPES pHは7.9、250 mMのMgCl 2）2 X結合および洗浄（B＆W）緩衝液（10mMトリス（pH7.5）、1mMのEDTA、2MのNaCl、0.02％Tween 20）で。 </td></tr></tbody></table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5 - メチルシトシン（5-MC）は、ヒトのゲノムDNAの総シトシン〜2から8パーセントを構成しており、遺伝子発現、ゲノムの完全性の維持、親の刷り込み、X染色体不活性化の調節を含む生物学的機能の広い範囲に影響を与え発達、老化、癌1。最近では、酸化された5-MC、5 -ヒドロキシメチル（5 - HMC）の存在は、胚性幹（ES）細胞および神経細胞2-4の、特に哺乳動物細胞内で発見されました。 5-HMCは、TETファミリー鉄（II）/α-ケトグルタル酸依存オキシゲナーゼ2、3、5-MC触媒作用の酸化によって生成されます。 5-HMCは胚性幹（MES）細胞、正常な造血及び悪性腫瘍、および受精卵開発2、5月10日の維持に関与することが提案されている。良い5-HMCの機能を理解するために、信頼性が高く、簡単なシーケンシングシステムが不可欠です。従来のバイサルファイトシークエンシングは5-MC 11から5-HMCを区別することはできません</sup&gt;。 5-HMCの生態を解明するために、我々は具体的には5-HMCに12グルコース部分を追加したバクテリオファージ酵素を利用して、ラベルと5-HMCをキャプチャするための非常に効率的かつ選択的な化学的なアプローチを開発しました。 ここでは5-HMCの選択的化学標識するための簡単​​な2ステップの手順を説明します。第一の標識のステップでは、ゲノムDNA中の5 - HMCは、5 - HMCに6 - アジド-グルコースを転送する方法では、β-GTは、バクテリオファージT4からグルコシルトランスフェラーゼによる6 - アジド-グルコースで触媒で標識されている修正された補因子は、UDP-6-N3-GLC（6 N3UDPG）。第二段階、ビオチン化では、ジスルフィドビオチンリンカーをクリックケミストリーによりアジド基に装着されている。両方の手順は、ゲノム領域における5-HMCの豊富さに関わらず、ラベリングと非常に低いバックグラウンドを与えて完成につながる、非常に特異的かつ効率的である。 5-HMCのビオチン化に続いて、5-HMCを含むDNA断片を選択的に捕獲される密度に依存しない方法でストレプトアビジンビーズを使用。得られた5-HMC-濃縮したDNA断片を、次世代シーケンシングを含む下流の分析、を使用することができる。 私たちの選択的標識とキャプチャプロトコルは、変数/多様な5-HMCの存在量を用いたゲノムDNAの任意のソースに適用し、高感度を与える。このプロトコルの主な目的は、その下流のアプリケーション（ すなわち 。、次世代シーケンシングは、ゲノム中の5-HMC分布をマップアウトする）ですが、それは単一分子であり、リアルタイムSMRT（DNA）の配列決定、と互換性があります5-HMCの単一塩基解像度のシーケンシングを提供することができる。

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

説明は簡単で、密度に依存しない濃縮のためのビオチンリンカーを転送するために、クリックケミストリー、続いて5-HMCにアジド-グルコースを転送するために、β-グルコシルトランスフェラーゼ（β-GT）を用いた二段階ラベリングプロセスである。この効率的で特異的標識法は、次世代シーケンシングを介して非常に低いバックグラウンドと高いスループットepigenomicマッピングで5-HMCの濃縮を可能にします。

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 ...

selective-capture-of-5-hydroxymethylcytosine-from-genomic-dna

Research

JoVE Journal

Biology

11.9K ビュー.  Emory University School of Medicine. 説明は簡単で、密度に依存しない濃縮のためのビオチンリンカーを転送するために、クリックケミストリー、続いて5-HMCにアジド-グルコースを転送するために、β-グルコシルトランスフェラーゼ（β-GT）を用いた二段階ラベリングプロセスである。この効率的で特異的標識法は、次世代シーケンシングを介して非常に低いバックグラウンドと高いスループ...

ビデオ: Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

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

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 ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

土壌と堆積物から高分子量のゲノムDNAの抽出

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

プライマー伸長キャプチャ：重縮退DNAソースからターゲットシーケンスの取得

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

RNAを含むオリゴヌクレオチドを​​使用して変換によるゲノムDNAのRNA / DNAハイブリッドの世代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

原核生物培養液からプラスミドDNAの合理化精製

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

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 ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

バイオマットから高分子量DNAの抽出

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

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 ...