科学と芸術のクロアチアアカデミーとロジスティックと科学的サポートのための科学のベルリン - ブランデンブルクアカデミー;と財政支援のためのマックスプランク協会の大統領イノベーション基金私たちは、アドバイスM.マイヤーに感謝。 JMGは、NSFの国際ポスドク（OISE - 0754461）によってサポートされていました。ネアンデルタール人の1（Feldhofer 1）FM865407、ネアンデルタール人2（Feldhofer 2）FM865408、Sidron 1253 FM865409、Vindija 33.25 FM865410、Mezmaiskaya 1 FM865411：シーケンスは、アクセッション番号とEBIヌクレオチドデータベースに寄託されています

このメソッドは、で報告された研究で使用されて<a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">ブリッグスら、Science 325（5938）。 318〜321（2009） 。</a> 必要な試薬： <ul><li>のAmpliTaqゴールドDNAポリメラーゼ</li><li> 10倍ジーンアンプPCRバッファーII </li><li> MgCl 2の 25mMの</li><li> dNTPミックス、25mMの各</li><li> BSA、10 mgの水のML - 1 </li><li>分子生物学グレードの水</li><li> EBバッファ（アプライPCR精製キットに付属）、10mMのトリスHCl、pH8.5で</li><li>アプライPCR精製キット</li><li> M - 270ストレプトアビジンのDynabeads </li><li> 2倍のバインドとウォッシュ（BW）バッファー（2 M NaCl、10mMのトリス- Cl、1mMのEDTA、pH8.0の0.2％ツイーン20） </li><li> 1Xバインディングとウォッシュ（BW）バッファー（2 × BindWashのバッファ水で2倍希釈） </li><li>ホットウォッシュ（HW）バッファ（2.5mmの塩化マグネシウム、1 × Taqポリメラーゼゴールドバッファー、0.1％のTween 20） </li></ul>非標準試薬と機器に関するメーカーの情報については、後でテーブルを参照してください。 1）図書館の増幅<ol><li>ここにDNA鋳型は454で説明されているように、低コピー数のDNAの源から調製したライブラリ（RohlandとHofreiter、2007A、2007B）と（MaricicとPääbo、2009）である。ライブラリ全体を増幅するために、以下のPCRミックスを調製： <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 試薬 </td><td> 反応あたりμL </td></tr><tr><td>水</td><td> 45 </td></tr><tr><td> 10XジーンアンプPCR緩衝液II </td><td> 10 </td></tr><tr><td>のMgCl 2（25mMの） </td><td> 10 </td></tr><tr><td> BSA（10 mg / ml）を</td><td> 2 </td></tr><tr><td>のdNTP（25mMの各） </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCRのRV車のprimer/10uM </td><td> 3 </td></tr><tr><td>のAmpliTaqゴールドのDNAポリメラーゼ（5 U /μL） </td><td> 1 </td></tr><tr><td> 454 DNAライブラリーのテンプレート</td><td> 25 </td></tr><tr><td> 合計 </td><td> 100 </td></tr></tbody></table></li><li> 14サイクルの増幅のためのサーモサイクラーに以下のPCRプログラムを使用して、 <ol><li> 95℃12分</li><li> 95℃30秒</li><li> 60 ° C（または、目的のアニーリング温度）1分</li><li> 72℃1分</li><li> 2（× 13）に移動します</li><li> 72℃5分</li><li> 10 ° C∞ </li></ol></li><li>製造元の指示に従ってキアゲンMinEluteスピンカラム上の反応を清める。 50μlのバッファーのEBで溶出します。 </li><li>精製された増幅産物だけでなく、定量PCRで増幅されていないライブラリのアリコート（Meyerら、2007）定量化する。増幅産物は、ULあたり以上1 × 10 12個のコピーを持っている場合ではなく、2つ以上のX 10 12コピーは、プライマー伸長反応に追加されるように以下のプライマー伸長のステップでテンプレートの量を減らす。 </li></ol> 2）プライマー伸長<ol><li>反応の必要な数のマスターミックスを調製する。 <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> 試薬 </td><td> 反応あたりμL </td></tr><tr><td>水</td><td> 45 </td></tr><tr><td> 10XジーンアンプPCR緩衝液II </td><td> 10 </td></tr><tr><td>のMgCl 2（25mMの） </td><td> 10 </td></tr><tr><td> BSA（10 mg / ml）を</td><td> 2 </td></tr><tr><td>のdNTP（25mMの各） </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCRのRV車のprimer/10uM </td><td> 3 </td></tr><tr><td>のAmpliTaqゴールドのDNAポリメラーゼ（5 U /μL） </td><td> 1 </td></tr><tr><td> 454 DNAライブラリーのテンプレート</td><td> 25 </td></tr><tr><td> 合計 </td><td> 100 </td></tr></tbody></table></li><li> 単一のプライマー伸長反応のためのサーモで次のプログラムを実行します。 <ol><li> 95℃12分</li><li> 60 ° C（または、PECのプライマーのアニーリング温度と異なる場合）1分</li><li> 72℃5分</li><li> 72 ° C永遠に！ 重要なステップは、72で拡張後の反応℃以下でご使用ください。ヒートブロックから削除する前に、チューブにQIAGENアプライの​​精製のため、直接ピペット150ul PBIまたはPBバッファ。これは、アニーリング非特異的プライマーを避けるために、混合物は冷却としてキャプチャすることが重要です。 </li></ol></li><li>製造元の指示に従って、キアゲンMinEluteスピンカラム上に反応して浄化する。 50μlのEBで溶出。 </li></ol> 3）拡張製品のキャプチャ<ol><li>ボルテックスによりM - 270ビーズの再懸濁し、ストック溶液。サンプルあたり25μlのビーズ懸濁液を取り出してください。サンプルあたり25μlの2xBWバッファに500ul 2倍BWバッファーに懸濁するで二回ビーズを洗浄してください。 </li><li>ステップ3.1からステップ2.3から25μlのビーズ懸濁液に25μlの溶出液を追加。 1に対して回転して混ぜる室温で5分。 推奨 ：定量PCRの定量化のためのステップ2.3からの溶出液の5μlの残り続ける。 </li><li>新鮮な1.5mlチューブに混合物全体を転送する（これは、非ターゲットライブラリのフラグメントのキャリーオーバーを減らすのに役立ちます）。ペレット磁気粒子のコレクタ（MPC）を用いて上清、上清を捨てる。 </li><li> 500μlの1xBWバッファー（多くの洗浄はできるだけ多くのバックグラウンドとして除去するのに役立つ）で5回洗浄する。最後の洗浄ステップの後、一時的にスピンダウンし、上清の最後の痕跡を削除する。 </li><li> 500μL× 1ホットウォッシュ（HW）バッファを追加します。サーマルブロックに65 ° C（またはPECプライマーのTm上記5C）で2分間振とうする。冷却最小限に抑えるために、この後すぐに上清を取り除く。 （このステップはまだPECプライマーではなく、実際に延長工程の間に延長に関連付けられている背景の断片を削除することです）。上清の最後の痕跡を削除します。 </li><li> 30μlのEBバッファにビーズペレットを再懸濁する。新鮮な1.5mlチューブに混合物全体を転送する（これは、非ターゲットライブラリのフラグメントのキャリーオーバーを減らすのに役立ちます）。溶出用サーマルサイクラーで3分間95℃でインキュベートする。 MPCの場所と背後にあるビーズを残すように注意しながら、上清を取り除く。 </li></ol> 4）製品の増幅をキャプチャ<ol><li>サンプルの必要な数のPCRマスターミックスを調製する。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 試薬 </td><td> 反応あたりμL </td></tr><tr><td>水</td><td> 45 </td></tr><tr><td> 10XジーンアンプPCR緩衝液II </td><td> 10 </td></tr><tr><td>のMgCl 2（25mMの） </td><td> 10 </td></tr><tr><td> BSA（10 mg / ml）を</td><td> 2 </td></tr><tr><td>のdNTP（25mMの各） </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCRのRV車のprimer/10uM </td><td> 3 </td></tr><tr><td>のAmpliTaqゴールドのDNAポリメラーゼ（5 U /μL） </td><td> 1 </td></tr><tr><td>溶出されたキャプチャ製品</td><td> 25 </td></tr><tr><td> 合計 </td><td> 100 </td></tr></tbody></table></li><li> 14サイクルの増幅のためにサーモサイクラーでは、次のPCRプログラムを使用します。 <ol><li> 95℃12分</li><li> 95℃30秒</li><li> 60 ° C（または、目的のアニーリング温度）1分</li><li> 72℃1分</li><li> 2（× 13）に移動します</li><li> 72℃5分</li><li> 10 ° C∞ </li></ol></li><li>製造元の指示に従ってキアゲンアプライシリカスピンカラムを介して反応を清める。 50μlのEBのバッファーで溶出。製品はステップ2.1で始まる、キャプチャの第二ラウンドのいずれかを使用、または順序付けのために、454エマルジョンPCRのプロトコルに直接入力できます。 </li></ol>代表的な結果： 推奨ノーマルと混在さ - &quot;陽性対照標的配列&quot;（1ピコグラムは、簡単に十分な例：a〜100ベーシスポイントPCR産物）の少量が最初に捕獲反応を行うためにPECのプロトコルを使い始めたときにそれを強くお勧めします。増幅された454ライブラリのテンプレート（ステップ2.1参照）の量、およびキャプチャはポジティブコントロールの製品をキャプチャするために設計された単一のPECのプライマーを使用して実行されます。このコントロール反応が行われる場合、ポジティブコントロール固有の、454アダプタ固有のプライマーペアの両方の定量PCRは、精製したプライマー伸長反応（1.3）で&quot;制御対象&quot;対バックグラウンドの量を定量することができる、プライマー伸長生成物（2.3 ）と溶出ビーズキャプチャ製品（3.6）。この方法では、あなたの実験条件におけるバックグラウンドの除去（&quot;特異性&quot;）とターゲットの回復（&quot;感度&quot;）の効率性の両方の効果を直接測定することができます。 成功したPECプロト​​コルは、通常、次のプロパティがあります - 感度（プロトコルを介して保持された制御対象の量によって測定される）： 溶出ビーズキャプチャ製品で制御対象の合計金額（3.6）=〜プライマー伸長反応（2.3）精製における総量1〜20％。 背景 （454 emPCRのプライマー対によって測定される）： 溶出ビーズキャプチャ製品（3.6）で背景の総量&lt;精製したプライマー伸長反応（2.3）の合計額の0.01％。 最終製品に残っているターゲットの1％未満がある場合は、プライマー伸長のステップは失敗している可能性があり、調査する必要があります。 最終製品に残っている背景の0.01％よりもはるかにある場合は、洗浄ステップが正しく実行された可能性があり、調査する必要があります。

<ol>
	<li>Briggs, A. W., Good, J. M., Green, R. E., Krause, J., Maricic, T., Stenzel, U., Lalueza-Fox, C., Rudan, P., Brajkovic, D., Kucan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+retrieval+and+analysis+of+five+Neandertal+mtDNA+genomes.">Targeted retrieval and analysis of five Neandertal mtDNA genomes.</a> Science. 325 (5938), 318-321 (2009).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+optimization+of+ancient+DNA+extraction.">Comparison and optimization of ancient DNA extraction.</a> Biotechniques. 42 (3), 343-352 (2007).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+DNA+extraction+from+bones+and+teeth.">Ancient DNA extraction from bones and teeth.</a> Nat Protoc. 2 (7), 1756-1762 (2007).</li><li>Maricic, T., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+454+sequencing+library+preparation+from+small+amounts+of+DNA+permits+sequence+determination+of+both+DNA+strands.">Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.</a> Biotechniques. 46 (1), 51-57 (2009).</li><li>Meyer, M., AW, B. r. i. g. g. s., Maricic, T., Hober, B., Hoffner, B., Krause, J., Weihmann, A., Paabo, S., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+micrograms+to+picograms:+quantitative+PCR+reduces+the+material+demands+of+high-throughput+sequencing.">From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.</a> Nucleic Acids Res. 36 (1), e5-e5 (2007).</li></ol>

The authors have nothing to disclose.

PECの方法は、簡単、迅速、高感度かつ特異的である。したがって、我々は、著者などのRNAライブラリから小さなRNA断片のキャプチャのような古代のDNAの外側に描く、複数のアプリケーション、メタゲノムサンプルからプールされたサンプルや16Sのキャプチャ（または他の遺伝子座）多様性の構造変化の尋問。言及する一つのポイントは、キャプチャの感度は、多重捕獲反応が増加するのPECプライマーの数として低くなるということです。 PECは、したがって、理想的に（例えばメガベース以上）非常に大規模な捕獲領域のキャプチャには適していない、しかし非常によく迅速な方法で多くの個人から小規模なターゲット領域、あるいはSNP位置の捕獲に適しています。

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<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8080241</td>
<td>&nbsp;</td>
<tr>
<td>QIAGEN MinElute PCR purification kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28004</td>
<td>&nbsp;</td>
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<td>M-270 streptavidin beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
<td>&nbsp;</td>
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<td>Tween-20</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
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<td>DynaMag-2 magnetic particle separator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

古代の骨の典型的なように我々は、大きく劣化し、微生物のDNAで汚染されているDNAの源から標的DNA配列の取得の方法を提示する。方法は、大幅にPCRまたはショットガンシーケンシングのアプローチを指示するために相対的なサンプルの破壊とシーケンシング要求を減少させる。我々は、地理的範囲にわたってから五ネアンデルタール人の完全なミトコンドリアDNA（mtDNA）のゲノムを再構成するためにこのメソッドを使用する。後期ネアンデルタール人のミトコンドリアDNAの遺伝的多様性は、現代的な現代人のそれよりも約3倍低かった。一緒にミトコンドリアのタンパク質の進化の分析と、これらのデータは、ネアンデルタール人の長期的効果的な集団サイズが現代人と現存の大型類人猿のそれより小さかったことを示唆している。

Watch this Scientific Journal Video about Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources at JoVE.com

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

我々は、我々は5つのネアンデルタール人の完全なミトコンドリアゲノムを再構築するために使用されるターゲット古代のDNA配列の検索、の方法を提示する。現代の人間とのこれらの配列の比較は、ネアンデルタール人は、長期の低効果的な人口の大きさを持っていたことを示唆している。

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 ...

primer-extension-capture-targeted-sequence-retrieval-from-heavily

Research

JoVE Journal

Biology

20.0K ビュー.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. 我々は、我々は5つのネアンデルタール人の完全なミトコンドリアゲノムを再構築するために使用されるターゲット古代のDNA配列の検索、の方法を提示する。現代の人間とのこれらの配列の比較は、ネアンデルタール人は、長期の低効果的な人口の大きさを持っていたことを示唆している。

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

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

マウス卵母細胞の検索

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

生物学

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

ショウジョウバエにおけるテングザル拡張レスポンス（PER）アッセイ

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Isolation of Genomic DNA from Mouse Tails

マウスの尾からのゲノムDNAの単離

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

ランダムDNAライブラリーを使用するプライマーフリーアプタマーの選択

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

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 ...

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 ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

転写開始点を決定するための蛍光に基づくプライマー伸長技術とのRNaseの切断サイト<em&gt;インビボ</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

次世代シーケンシングによりDNAメチル化解析をターゲット

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

迅速な免疫標識と合わせてレーザーキャプチャーマイクロダイセクションを使用して、細胞特異的な亜集団からのRNAの単離

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...