Name: an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants
Uploaded: 2017-11-08T15:00:00
Description: Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

この作品が資金を提供一部助成金 NSF 植物ゲノム研究 (IOS 1127155) から。

注: 図 1 アレイ CGH のプロトコルの 5 つの手順を示しています。彼らは、: 1) 工場用資材の準備2) 高品質 DNA サンプルの分離3) ラベリングと DNA サンプルの精製4) ハイブリダイゼーション、洗浄、および全ゲノム配列のスキャン・ 5) CGH データ分析。M. 糸状菌 ゲノム タイリング アレイには、50,000 以上の遺伝子または遺伝子ゲノム (材料の表 を参照) モ...

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アレイ ・ ベース CGH プラットフォームの検出と高速中性子照射 (FNB) の特性を開発した- M. 分子品種 Jemalong A17 の突然変異体の誘発。アレイ CGH 法遺伝子変異の検出の使用を示すためには、aCGH S. 法 Sm1021を接種したときに野生型植物と対照をなしてハイパー根粒形成表現型を表わした FN6191 変異体の解析を行った。セグメント化の分析のため、セグメントはセグメント内プローブは...

マメ科植物 (マメ科)、大豆 (ダイズ) やアルファルファ (アルファルファ) など多くの経済的に重要な種の顕花植物の 3 番目に大きい家族です。マメ科植物は根粒が大気中の窒素をアンモニア用にホスト植物によって減少を開発する根粒菌の総称で、土壌窒素固定細菌と対話できます。など、マメ科作物の栽培は窒素肥料の少しの入力を必要とし、持続可能な農業に貢献。葉と種子と高たんぱく、優秀な牧草と穀物マメ科作物を生成します。ただし、栽培されているマメ科草種は一般的にマメ科特有のプロセスが面倒で重要な役割を果たす遺伝子の機能解析を行う複雑なゲノム構造を持ちます。ウマゴヤシ分子採用されているマメ科植物の研究のためのモデル種として主な理由は (1) がある比較的小さい半数体ゲノムのサイズ (~ 550 Mbp); 二倍体ゲノム(遺伝子機能研究のため 2) 植物を安定して変換できます。(3) はアルファルファ (M. サティバ)、牧草、女王とトランスレーショナル研究の他の多くの経済的に重要な穀物に密接に関連。最近、ゲノム Jemalong A17 M. 分子cv のリリース1,2をされています。ゲノムの注釈は、50,000 以上の予測遺伝子やゲノムの遺伝子モデルがあることを示しています。M. 分子の遺伝子のほとんどの機能を決定するには、ゲノムはやりがいのある仕事です。遺伝子の機能解析を容易にするために以上 150,000 M1行の範囲の突然変異体の包括的なコレクションによって生成された高速中性子照射 (FNB) 突然変異誘発M. 分子cv Jemalong A173 、4。シロイヌナズナ5,6イネ (イネ)7トマト (ナスを含む多くの植物種の突然変異体の生成に高速中性子、高エネルギー イオン化変異原が使用されていますlycopersicum)、大豆 (ツルマメ;ミヤコグサ大麦 (オオムギ)、 G. 最大)8,910.FNB 変異から派生した突然変異の大部分は、メガ塩基対9,11に数塩基対からサイズの範囲を DNA 削除原因です。多くの表現型に関連する遺伝子が正常にされている識別し、特徴付けられる4,12,13,14,15,16,17,18,19. 以前は、地図ベースのアプローチは時間がかかるし、分子レベルで特徴付けられる突然変異体の数を制限に頼って FNB 変異体から根本的な遺伝子の分子クローニングします。最近では、トラン スクリプト ベースのメソッドを含むいくつかの無料のアプローチ、ゲノム DNA コピー数変化検出、配列する全ゲノムの配列に基づく比較 genomic の交配 (CGH) をタイル採用されている容易にするために、動物や植物20,21,22,23,24,25を含む多様な生物の欠失変異株の解析 26,27,28,29,30,31。
FNB M. 分子、全ゲノム配列に基づく比較 genomic の交配 (CGH) 突然変異体の解析を容易にするには、プラットフォームを開発し、検証されています。動物システムで報告は、CGH アレイ ・ ベース プラットフォームはコピー数変化 (CNVs) M. 分子FNB 変異体のゲノム全体レベルでの検出をことができます。さらに、PCR で病変が確認でき、罫線削除は、シーケンスによって識別できます。全体的にみて、アレイ CGH プラットフォーム、 M. 分子FNB 変異体の病変を識別するに効率的かつ効果的なツールです。ここでは、アレイ CGH プロシージャおよび削除ボーダー M. 分子FNB 変異株の PCR 解析を示します。
次のプロトコルは、コピー数の全ゲノム配列に基づく比較 genomic の交配 (CGH) 解析を行うに興味がある人のため実験手順、試薬、装置および分析ツールに関する情報を提供します。植物の変化。例として、ウマゴヤシ分子FN6191 変異体は、削除領域および突然変異体の表現型に関連する候補遺伝子を識別するために使用されました。M. 分子FN6191 変異体は、もともと由来の高速中性子照射誘起削除変異コレクション32 (材料の表を参照)、土と接種後のハイパー根粒形成表現型の展示細菌、 Sihorhizobium 分類Sm1021、野生型植物とは対照的。

<table>
	<tbody>
		<tr>
			<td>Medicago truncatula genome array, 1 x 1 M</td>
			<td>Agilent</td>
			<td>G4123A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula FN6191 (mutant)</td>
			<td>In house</td>
			<td>FN6191</td>
			<td></td>
		</tr>
		<tr>
			<td>Medicago truncatula Jemalong A17 (reference)</td>
			<td>In house</td>
			<td>A17</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>320501</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Plant Mini Kit</td>
			<td>Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>1000D</td>
			<td></td>
		</tr>
		<tr>
			<td>SureTag DNA Labeling Kit</td>
			<td>Agilent</td>
			<td>5190-3400</td>
			<td></td>
		</tr>
		<tr>
			<td>Random primer</td>
			<td>Agilent</td>
			<td>5190-3399</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>MJ research</td>
			<td>PTC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Labnet international Inc</td>
			<td>Spectrafuge 24D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stabilization and Drying Solution</td>
			<td>Agilent</td>
			<td>5185-5979</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Hybridization Kit</td>
			<td>Agilent</td>
			<td>5188-5380</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber gasket slides</td>
			<td>Agilent</td>
			<td>G2505</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Agilent</td>
			<td>5190-3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo aCGH/ChIP-on-chip Wash Buffer 1 and 2</td>
			<td>Agilent</td>
			<td>5188-5221</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Chamber, stainless</td>
			<td>Agilent</td>
			<td>G2534A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Agilent</td>
			<td>G2545A</td>
			<td></td>
		</tr>
		<tr>
			<td>Purification Columns</td>
			<td>Agilent</td>
			<td>5190-3391</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanner</td>
			<td>Roche</td>
			<td>MS200</td>
			<td></td>
		</tr>
		<tr>
			<td>NimbleScan 2.6</td>
			<td>Roche Nimblegen</td>
			<td>5225035001</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal Map 1.9</td>
			<td>Roche Nimblegen</td>
			<td>Signalmap1.9</td>
			<td></td>
		</tr>
	</tbody>
</table>

an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants

変異体は、遺伝子機能研究のための非常に貴重な遺伝資源です。突然変異体のコレクションを生成するため、T-DNA やトランスポゾンなどの生物、メタンスルホン酸エチル (EMS) などの化学物質や電離放射線などの物理など、変異原物質の 3 つのタイプを利用できます。突然変異の観察の種類使用変異原によって異なります。電離放射線誘発突然変異の突然変異には、削除、複製、または再配置が含まれます。T DNA やトランスポゾンを用いた突然変異誘発は変換に影響を受けやすい種に限られているが、化学的または物理的な突然変異は種の広い範囲に適用できます。しかし、伝統的に化学的または物理的な突然変異から派生した突然変異の特性は、マップベースの複製のアプローチ、集中的な時間のかかる労働をあるに依存します。ここでは、効率的に検出し、コピー数変化 (CNVs) 変異株由来の高速中性子照射 (FNB) 突然変異を特徴付ける高密度のゲノム配列に基づく比較 genomic の交配 (aCGH) プラットフォームを適用できることを示すウマゴヤシ分子、マメ科草種。全ゲノム シーケンス解析は、50,000 以上の遺伝子または遺伝子M. 分子モデルがあることを示しています。M. 分子の存在、FNB 誘発突然変異体でゲノムにおける遺伝子機能研究のための非常に貴重な遺伝資源を表す以上 150,000 の M1 線から派生されます。ここで説明した aCGH プラットフォームは、 M. 分子で FNB 誘発突然変異体を特徴付けるための効率的なツールです。

図 2は、全ゲノムを渡る WT 信号対変異体の正規化 log2比の分布を示しています。CGH のデータの分析はおおよそ明らかになった全体サンヘンプ遺伝子33と他のいくつかを含む, 染色体 4 22 kb 削除注釈遺伝子の FN6191 変異体 (図 2、図 3)。候補削除された領域は、-2.5 (?...

Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

このプロトコルは、コピー数変化の全ゲノム配列に基づく比較 genomic の交配 (CGH) 解析を行うに興味を持っている研究者の実験の手順と分析ツール、機器、試薬に関する情報を提供します。植物。

アレイ ・ ベース比較 Genomic 交配用高速中性子誘起ウマゴヤシ分子変異体のコピー数変化の効率的な検出

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

The overall goal of this array-based comparative genomic hybridization protocol is to rapidly identify copy number variations in fast neutron bombardment induced mutants of the legume plant Medicago truncatula. This method helps to answer key questions in the field of legume biology. Such as, facilitating identification of empathogens on low site involved in regulating nodule development in legumes.The main advantage of this technique is that it is of highest repute and sensitive in detecting copy number variations such as deletion mutations in the neutron bombardment mutants of the legume plant Medicago truncatula. Demonstrating this procedure will be myself, and Hongcheng Wang, Host Doctor Fellows from Genetic Lab. Quickly freeze one gram of young leaf tissue collected from single plants in liquid nitrogen.Then, use a mortar and pestle and grind the frozen leaf tissue to fine powder within the liquid nitrogen. Extract the wild type and mutant genomic DNA samples from the fine powder using a DNA isolation kit. Use 500 microliter centrifuge tubes to dilute one microgram of each wild type and mutant genomic DNAs with double distilled water to a final volume of 20 microliters.Then, add five microliters of random primer to the centrifuge tubes. Quickly vortex the tubes, and after a quick spin down place them in a thermocycler for 10 minutes to denature the DNA samples at 98 degrees Celsius. After 10 minutes remove the tubes from the thermocycler, and instantly place them on ice water for five minutes.Then, prepare two labeling mixes and add 25 microliters of labeling mix as one and two to the wild type and mutant DNA tubes respectively. Pipette the DNA and the labeling mix three times, and then briefly spin the tubes. After spinning, incubate the tubes in the thermocycler at 37 degrees Celsius for two hours, and then at 65 degrees Celsius for 20 minutes to inactivate the exo Klenow enzyme.Then, add 430 microliters of One X TE Buffer, and mix the contents in each tube. Next, briefly spin the tubes and transfer the solution in the tubes to purification columns with two milliliter collection tubes. Centrifuge the purification columns at 14, 000 times G for 10 minutes and discard the flow through.Add 480 microliters of One X TE Buffer to each column, and again centrifuge at 14, 000 times G for 10 minutes. Discard the flow through. Transfer each of the wild type and mutant labeled DNAs from the bottom of the column to new centrifuge tubes.After measuring the volume of each of the labeled DNAs with the pipette adjust the final volume to 80 microliters with One X TE Buffer, then measure the concentration of the labeled DNAs in a spectrophotometer. Next, mix equivalent volumes of mutant and wild type DNAs to hybridize probes on a micro array chip for comparative hybridization. Bring the final volume to 160 microliters with One X TE Buffer.Next, prepare the hybridization solution and pipette it three times to mix three agents well with mixed DNA. After briefly spinning the tubes incubate them in a thermocycler at 98 degrees Celsius for 10 minutes, and at 37 degrees Celsius for 20 minutes. Remember to set the temperature to 67 degrees Celsius to preheat the hybridization oven four hours before hybridization.Then, place a gasket slide on the base of the hybridization chamber and load 490 microliters of the hybridization solution onto it after 20 minutes of incubation at 37 degrees Celsius in the thermocycler. To form the hybridization chamber cover the gasket slide with Medicago truncatula genome micro array chip, then cover the hybridization chamber and securely tighten it with the clamps. Incubate the assembled hybridization chamber in the hybridization oven at 67 degrees Celsius for 40 to 48 hours.Meanwhile, prepare two slide washing dishes with 250 milliliters of Washing Buffer One kept at room temperature. Then, make one slide washing dish with Washing Buffer Two kept at 37 degrees Celsius. Then, prepare one slide washing jar with 70 milliliters of acetonitrile, and another slide washing jar with 70 milliliters of stabilization and drawing solution.Place both the slide washing jars in a fume hood at room temperature. After incubation remove the hybridization chamber from the hybridization oven and loosen the chamber clamps to open the cover. Then, remove the micro array chip in the gasket slide and place them on slide washing dish with Wash Buffer One.Isolate the micro array chip from the cover slide. Next, transfer the micro array chip to the second slide washing dish with Wash Buffer One. Using a stir bar, gently stir the solution on a magnetic stir plate at room temperature for five minutes.Place the micro array chip on the slide washing dish with pre-warmed Washing Buffer Two, then stir with the stir bar to wash the solution at 37 degrees Celsius for one minute. Remove the micro array chip and place it in the acetonitrile containing slide washing jar in a fume hood to wash for 30 seconds. Transfer the micro array chip to the slide washing jar with stabilization and drying solution and wash again for 30 seconds.Carefully remove the chip and dry for one minute inside the fume hood. Scan the micro array chip using a scanner under two micrometer resolutions. A signal mapping software was used as an interface to analyze the distribution of normalized log two ratios of mutant versus wild type signals across eight chromosomes.For putative deletions value of log two ratio equal to or less than minus two point five standard deviation is considered. Segmentation analysis of the software demonstrates an estimated 22 kilobase deletion region on chromosome four shown in the graph. Analysis of the deletion borders flanked by the micro array probes show a reduced mean normalized log two ratio.The graph also shows six other annotated genes including the son gene encompassing the deleted region. Son gene is responsible for controlling nodulation in Metecago truncatula. Next, PCR amplification done to confirm the deletion borders shows a one point five kilobase product amplified from FN6191 mutant, but not in the wild type.DNA sequencing was done to show the deletion junction in FN6191 mutant shown in the black arrow. Sequencing was also done that confirmed the location of the deletion borders. Once mastered, this technique can be completed in 72 hours if it is performed properly.To obtain high quality data, remember to keep the ozone concentration low in the room where performing the procedure. Following this procedure, other measures such as polymerase chain reactions of all genome sequencing can be performed to answer additional questions like the size and the copy number variations in the genome. Event and validation of this technique has paved the way for researchers to explore copy number variation they induce to mutants and the natural variants in plant species that are not amenable to insertional mutagenesis such as Garden P.Don't forget that working with acetonitrile, stabilization and drying solution can be extremely hazardous.Precautions, such as wearing eye proctection, avoiding direct contact with the agents, and working careful is absolutely essential.

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An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

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アレイ ・ ベース比較 Genomic 交配用高速中性子誘起ウマゴヤシ分子変異体のコピー数変化の効率的な検出

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