1。試料捕集<ol><li>適切なインフォームドコンセントを持つ個人からの血液サンプルを収集します。個々については、P100チューブ（BD診断、フランクリン湖、NY、USA）への血液4〜を5ml描く。比較のために、同時にEDTAチューブに血液を採取。 </li><li> BD P100管は15.8ミリリットル、噴霧乾燥K2EDTAおよび凝固を防止し、血漿タンパク質を安定化するプロテアーゼ阻害剤の凍結乾燥された独自の広域スペクトルカクテルを含有する。彼らはまた、機械的な区切り文字を含んでいます。処理が発生するまで、全血を採取した後、一晩、60分間室温で両方のチューブを保持する。 </li><li>室温で15分間、2500グラムでBD P100チューブを遠心する。マイクロ遠心チューブにメカニカルセパレーターの上に採取した血漿を転送します。 </li><li> -80℃で、セパレーターの下に圧縮血を含む、P100チューブを保管°Cあなたは、DNA内線を開始する準備が整うまでraction。 </li></ol> P100管の血（P100_DNA）から2.1 <ol><li> P100チューブを-80℃で保存するプラズマ抽出後のC。 3から4ヶ月以内に、5分（ 図1A）のための水浴中で37℃でフリーザーや解凍から圧縮された血液を含むP100チューブを取り出す。セパレーターの上に座って任意の残留プラズマを削除します。セパレーター上記P100チューブ（ 図1B）で- 17％ショ糖溶液（未発表データ社内でテスト勾配溶液）2 mlを加え。 2500グラムで20分間室温で遠心する、このプロセスは、チューブの頂部に機械的なセパレータをプッシュし、軟膜（ 図1C）を含む溶液の三層が得られる。手動steriledフック金属クリップ（ 図1D）を使用して区切り文字を抽出します。 </li><li>各チューブ（ 図1D）から機械的な区切り文字を取得した後、削除してdiscarDトップ蔗糖層。無菌の15ミリリットルコニカルチューブに、バフィーコート（BC）層を表す中間層を、転送します。 3ミリリットルの軟膜の量を増加させるためにリン酸緩衝生理食塩水（PBS）を追加します。メーカーの推奨に従ってキアゲンプロトコールとトラブル血キットの試薬を用いてバフィーコートからDNAを抽出し、2500グラムの遠心速度（代わりに2000グラムの）を除いて。 </li><li>赤血球（RBC）を溶解して除去するために、バフィーコートの3 mlにRBC溶解9mlのを追加します。インキュベーション中時折反転と10分間、各チューブを室温で数回とインキュベートを反転。 5分間2500グラムで、それぞれの混合をスピンダウンし、上清を捨てる。 15秒のために細胞溶解液と渦の3mlで各チューブの残りのペレットを扱う。 15秒のためのタンパク質の沈殿溶液と渦の1mlで溶解液を扱う。 </li><li> 5分間2500グラムで遠心し、含有する新鮮な15ミリリットルコニカルチューブに上清を移す100％イソプロパノールを3ml。 3分間2500グラムで新しいチューブを10回と遠心分離機を反転。上清を捨て、70％エタノール1 mlを加える。 DNAペレットを取り除くと洗浄するコニカルチューブを数回反転させる。 1分間2500グラムで遠心。ペレットを室温で3分間（逆さまにチューブを配置）するために乾燥させた後、37℃で補水液250mlでDNAを中断℃で10分間、プレラベル1.7ミリリットルマイクロチューブへの転送のためのC。 -80℃でチューブを保管°Cを使用するまで。 </li></ol> EDTA Ttubeの血（EDTA_DNA）から2.2 <ol><li>ルーチン免疫血液学試験のための全血をK 2 EDTAの10.8ミリグラムでスプレーコーティングし、-80℃で保存プラスチックvaccutainerチューブに集められ管は機械的な区切りが含まれていないため、DNA抽出は、機械的なセパレーター（2.1.1および2.1.2）を含む手順は含まれていません。 </li><li>血液貯蔵、DNの3から4ヶ月以内カワセミフレックス（サーモフィッシャーサイエンティフィック、ウォルサム、MA、USA）を使用して抽出される。これは、磁性粒子技術に基づく自動化DNA抽出システムであり、細胞溶解/ DNA結合、数回の洗浄工程およびDNAの溶出で構成されている。 </li><li> DNA抽出に使用されるキットはカワセミフレックス24 DNA血液キット（Qiagen、ドイツ）である。 </li></ol> 3。 DNA量と品質評価ゲノムDNAの量、品質および完全性をピコグリーン、分光法及び電気泳動を含む方法の組み合わせにより評価した。 <ol><li>ゲノムDNAの濃度は、ナノドロップし、ピコグリーン定量化によって決定される。 </li><li>純度は、光度計、分光光度計での吸光度比260/280の測定値から決定される。 </li><li>試料（500ngの）も、DNAの完全性（分解）を評価するために、1％アガロースゲル上にロードされている。 </li></ol> 4。 Genotypinグラムアッセイおよび品質管理<ol><li>五P100_DNAとそれに対応するEDTA_DNAサンプルをランダムにカスタムImmunochip（DNA免疫分析ビーズチップ）を使用して、さらなる分析のために選択される。 Immunochipは196524多型を含むのInfiniumジェノタイピングチップで、免疫遺伝学的研究の5のために設計されています。 </li><li>各サンプルについて、200ngのDNAが増幅され、断片化され、沈殿させ、適切なハイブリダイゼーション緩衝液に再懸濁した。 </li><li>変性した試料は、16時間の最低48℃でImmunochipsにハイブリダイズさせる。 </li><li>ハイブリダイゼーション後、Immunochipsは、一塩基伸長反応で染色のために処理される。 </li><li> immunochipsその後イルミナHiscanビーズアレイリーダーと正規ビーズ強度データはイルミナGenomeStudioソフトウェアにロードされ、遺伝子型に変換され、使用して結像される。遺伝子型はGenomeStudioのautocallingアルゴリズムを使用して呼び出されます。における品質管理コー​​ルレート&lt;95％と一塩基多型の除外（のSNP）をvolves。 </li><li> SNPコールレートはimmunochipアッセイ効率の尺度として使用される。これは、自動化された遺伝子型の割り当てを受信するのに十分な信号強度及び品質を実現チップ（各チップにエンコードされた196524個のSNP）に関するアッセイの総数の百分率として算出される。高品質のDNAは、（260/280の比が1.8以上、劣化の有無）immunochipアッセイに含まれるハップマップコントロールDNAと少なくとも同じコール·レートを達成するために期待されている。品質管理コールレート&lt;95％の各データセット内でのSNPの排除を伴う。 </li></ol>

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利害の衝突は宣言されていない。

多くの人間の病気は、遺伝的な原因があり、人間の病気の遺伝的基礎を探求することは増加高品質のDNAが求められます。遺伝学的研究に使用されるDNAの最も一般的な原因は、全血を抗凝固される。ほとんどの臨床検査室では、生化学的な血清学、および表現型アウトカム調査の最初のステップのようなウイルスのテストを実行するので、血液がしばしばプラズマチューブに集められる。血漿?...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>BD P100 tubes</td>
 <td>BD Diagnostics</td>
 <td>366448</td>
 </tr>
 <tr>
 <td>EDTA tubes</td>
 <td>BD Diagnostics</td>
 <td>367863</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378</td>
 </tr>
 <tr>
 <td>Paper clip</td>
 <td>Office product</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Corning</td>
 <td>430052</td>
 </tr>
 <tr>
 <td>Red blood cells (RBC)</td>
 <td>Qiagen</td>
 <td>158389 (kit)</td>
 </tr>
 <tr>
 <td>Phosphate Buffer Saline (PBS)</td>
 <td>Thermo Scientific</td>
 <td>SH30256.01</td>
 </tr>
 <tr>
 <td>BioSprint 96 DNA Blood Kit (48) </td>
 <td>Qiagen</td>
 <td>940054</td>
 </tr>
 <tr>
 <td>1.7 ml microtubes</td>
 <td>Axygen</td>
 <td>MCT-175-C</td>
 </tr>
 <tr>
 <td>Human Immuno DNA Analysis BeadChip Kit</td>
 <td>Illumina</td>
 <td>WG-352-1001</td>
 </tr>
 <tr>
 <td>Bead array reader</td>
 <td>Illumina</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>GenomeStudio Software</td>
 <td>Illumina</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

a practical and novel method to extract genomic dna from blood collection kits for plasma protein preservation

臨床検査は、血液の細胞または流体部分に行うことができる。別の採血管を使用することにより分析することができる血液（全血、血漿または血清）の一部を決定する。人間の疾患の遺伝的基礎を研究に携わる研究所は、ゲノム/遺伝子解析のためのDNAの供給源としてEDTA含有バキュテナーに収集凝固処理全血に依存しています。ほとんどの臨床検査室は、表現型のアウトカム調査の最初のステップとして、生化学的、血清学的およびウイルスのテストを実行するため、抗凝固処理血液もヘパリン含有チューブ（プラズマチューブ）に集められる。したがってDNAプラズマがゲノムおよびプロテオミクスデータの両方の同時並列分析のために必要とされる場合には、EDTAおよびヘパリンチューブの両方に血液を採取するのが通例である。血液は単一の管に収集し、血漿とDNAの両方の原因となることができれば、その方法は、既存のメタへ前進であると考えられるODS。プラズマ抽出後の圧縮された血液の使用は、このように処理された血液試料の量を最小化し、各患者から必要なサンプル数を減らすこと、ゲノムDNAの代替供給源を表す。これは、最終的に時間とリソースを節約できるでしょう。 血漿タンパク質保存用BD P100血液採取システムは、ヒト血液から良好なタンパク質バイオマーカーの発見およびプロテオミクス実験を可能にし、血液のタンパク質含有量を安定させるために、前の血漿または血清採血管1よりも改善された方法として作成された。 BD P100管は、15.8、噴霧乾燥K2EDTA mlの凝固を防止し、血漿タンパク質を安定化するプロテアーゼ阻害剤の凍結乾燥された独自の広域スペクトルカクテルを含有する。また、遠心分離後、血漿および細胞ペレット間の物理的障壁を提供する機械的なセパレータを含む。いくつかの方法は、凝固した血液のsaからDNAを抽出するために考案されているmples古いプラズマチューブ2-4に収集。これらのメソッドからの課題は、主にチューブ（ゲル·セパレータ）の内部セパレータの種類に関連付けられており、凝固した血液の回収が困難である、断片化や血栓を分散させるの不便さ、および分離ゲルによって血栓抽出の閉塞を含まれていた。 私たちは、新しいBD P100チューブに採取した血液からゲノムDNAを抽出し、浄化する最初の方法を提示する。私たちは、P100のチューブからEDTA管からそれにDNAサンプルの品質を比較。我々のアプローチは、シンプルかつ効率的である。 2）機械的なセパレータの除去は、スクロースとの組み合わせを使用して、採血のための機械的なセパレータをプラズマBD P100（BD診断、スパークス、MD、USA）管の1）を使用する：これは、次の4つの主な手順が含まれ無菌クリップ金属フック、3）及び4白血球を含むバフィーコート層の剥離）からのゲノムDNAの単離軟膜は、通常の商業的なDNA抽出キット又は類似の標準プロトコルを使用する。

DNAの品質評価と濃度測定 P100_DNAsの収率はEDTA_DNAsのもの（ 表1、表2）よりも有意に低かった。その吸光度比260/280の値で表されるDNAの純度はP100_DNAとEDTA_DNAサンプルセット（ 表1および2）の両方に類似している。光スミア（ 図2）によって示されるように、いくつかの劣化が、EDTA_DNA試料中に見られるが、DNAの品質は、サンプ?...

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A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

私たちは、血漿/血清学的検査のために採取した全血からゲノムDNAを単離する新たな方法を説明しています。血漿採取した後、圧縮された血液は、通常廃棄されます。当社の新規の方法は、既存の方法に比べて大幅な改善を表し、追加の血液を要求せず、単一のコレクションからのDNAやプラズマを利用できるようになります。

血漿タンパク保存用採血キットからゲノムDNAを抽出するために実用的で新手法

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

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JoVE Journal

Biology

41.9K ビュー.  Emory University School of Medicine and Children's Health Care of Atlanta. 私たちは、血漿/血清学的検査のために採取した全血からゲノムDNAを単離する新たな方法を説明しています。血漿採取した後、圧縮された血液は、通常廃棄されます。当社の新規の方法は、既存の方法に比べて大幅な改善を表し、追加の血液を要求せず、単一のコレクションからのDNAやプラズマを利用できるようになります。

ビデオ: A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

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

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

アメリカカブトガニ、カブトガニポリフェムスから採血

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

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 ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

磁気活性化細胞選別（LIMACS）を使用して、脂質小胞を介したアフィニティクロマトグラフィー：タンパク質脂質相互作用を解析する新手法

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

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 ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Nonexpertsための系統学の実用ガイド

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

キナーゼ阻害剤に対して耐性変異のスクリーニングおよび検証のための方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

アダルトゼブラフィッシュにおける血液検査のための反復採血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

レトロウイルス統合サイトの増幅、次世代シーケンシング、およびゲノムDNAのマッピング

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...