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

이 작품의 자금에서 제공 됩니다 일부 권한을 부여 하 여 NSF 식물 게놈 연구 (IOS-1127155).

참고: 그림 1 배열 CGH 프로토콜에 대 한 5 단계를 보여 줍니다. 그들은: 1); 식물 재료의 준비 2) 격리의 고품질 DNA 샘플; 3) 레이블 및 정화의 DNA 샘플; 4) 교 잡, 세척, 그리고 검색의 전체 게놈 배열; 그리고 5) CGH 데이터 분석. M. truncatula 전체 게놈 타일링 어레이 50000 개 이상의 유전자 나 게놈 (참조 자료의 테이블)에 유전자 모델을 대상으로 하는 971,041 독특?...

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Medicago truncatula Mutant Database Available from: <a target="_blank" href="https://medicago-mutant.noble.org/mutant/FNB.php">https://medicago-mutant.noble.org/mutant/FNB.php</a> (2017)</li><li>Burton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+with+the+next+generation+of+CGH+microarray.">SNP genotyping with the next generation of CGH microarray.</a> MLO Med Lab Obs. 45 (7), (2013).</li></ol>

어레이 기반 CGH 플랫폼 검색 및 빠른 중성자 사격 (FNB)의 특성에 대 한 개발 했습니다-cv. M. truncatula Jemalong A17에에서 돌연변이 유도. 배열 CGH 메서드 유전자 돌연변이 검출에서 사용을 보여 우리는 돌연변이 FN6191 S. meliloti Sm1021와함께 접종 때 야생 유형 식물, 달리 하이퍼 nodulation 형 전시의 aCGH 분석을 수행. 세분화 분석을 위해 세그먼트 로그2 비율의 의미는 세그먼트 내의 프?...

콩 (Fabaceae) 콩 (최대 글리신), 알 팔 파 (개자리속 sativa) 등 많은 경제적으로 중요 한 종 가진 꽃 식물의 3 번째로 큰 가족입니다. 콩과 식물 질소 담합 토양 박테리아, 일반적으로 Rhizobia 있는 대기 이질소 호스트 식물에 의해 사용 하기 위해 암모니아로 감소 된다 뿌리 혹을 개발 하 라는 작용할 수 있습니다. 이와 같이, 콩과 식물 작물의 재배는 질소 비료의 작은 입력 요구 하 고 따라서 지속 가능한 농업에 기여. 콩과 식물 작물 생산 나뭇잎과 씨앗 높은 단백질 콘텐츠, 우수한 사료 및 곡물 작물으로 봉사. 그러나, 재배 콩과 식물 종에는 일반적으로 복잡 한 게놈 구조, 성가신 콩과 식물-특정 프로세스에 핵심 역할을 하는 유전자의 기능 연구를 만드는 있다. 개자리속 truncatula 널리 채택 되었습니다 콩과 식물 연구에 대 한 모델 종으로 주로 하기 때문에 (1)는 상대적으로 작은 단일 게놈 크기 (550 ~ Mbp); 2 중 게놈 (2) 식물 유전자 기능 연구; 대 한 안정적으로 변형 될 수 있다 그리고 (3)은 밀접 하 게 관련 된 알 팔 파 (M. sativa로 구나), 사료의 여왕 및 변환 연구에 대 한 다른 많은 경제적으로 중요 한 작물. 최근, 게놈 시퀀스 M. truncatula cv의 Jemalong A17 발표1,2되었습니다. 게놈 주석 50000 개 이상의 예측된 유전자 나 게놈에 있는 유전자 모델을 보여 줍니다. M. truncatula 에 유전자의 대부분의 기능을 확인 하려면 게놈은 어려운 작업입니다. 유전자의 기능 연구를 촉진 하기 위해1 라인의 범위에서 돌연변이의 포괄적인 컬렉션 생성 된 빠른 중성자 사격 (FNB) mutagenesis M. truncatula 이력서에 사용 하 여 Jemalong A173 4. 빠른 중성자, 고 에너지 이온화 돌연 애기5,6, 쌀 (Oryza sativa)7, 토마토 (푸른 풀밭을 포함 하 여 많은 식물 종에 돌연변이 생성에 사용 되었습니다. lycopersicum), 콩 (글리신 soja; 최대 G.)8,9, 보 리 (Hordeum vulgare), 그리고 로터스 나무10. FNB mutagenesis에서 파생 된 변이의 큰 부분 DNA 삭제 메가 기본적인 쌍9,11몇 가지 기본적인 쌍 크기에서 범위는 한다. 많은 표현 형 관련 유전자 성공적으로 되었습니다 식별 및 특징4,12,13,,1415,16, 17 , 18 , 19. 이전, 지도 기반 접근 시간이 소모 하 고 분자 수준에서 특징을 돌연변이의 수를 제한에 의존 FNB 돌연변이에서 기본 유전자의 분자 클로닝. 최근에, 몇 가지 무료 방법을 사본 기반 메서드를 포함 하 여, 게놈 DNA 복사 번호 유사 검색, 및 전체 게놈 시퀀싱, 배열 기반 비교 genomic 교 잡 (CGH) 기와 고용 촉진 하는 동물과 식물20,21,22,23,,2425을 포함 한 다양 한 유기 체에서 삭제 돌연변이의 특성 26,,2728,29,30,31.
M. truncatula는 전체 게놈 배열 기반 비교 genomic 교 잡 (CGH)에서 FNB 돌연변이의 특성화를 촉진 하기 위하여 플랫폼 개발 되어과 검증. 동물 시스템에 보고, CGH 배열 기반 플랫폼 M. truncatula FNB 돌연변이에 전체 게놈 수준에서 복사 번호 유사 (CNVs)의 탐지를 허용 한다. 또한, 병 변 PCR에 의해 확인 될 수 있습니다 및 삭제 테두리 시퀀싱에 의해 확인 될 수 있다. 전반적으로, 배열 CGH 플랫폼 M. truncatula FNB 돌연변이에 병 변 식별에 효율적이 고 효과적인 도구입니다. 여기, 배열 CGH 절차 및 PCR 특성화 M. truncatula FNB 돌연변이에 삭제 테두리의 그림은.
다음 프로토콜 복사 번호의 전체 게놈 배열 기반 비교 genomic 교 잡 (CGH) 분석 수행에 관심이 있는 연구자에 대 한 실험 단계 및 시 약, 장비 및 분석 도구에 대 한 정보 제공 유사 식물에서. 예를 들어, 개자리속 truncatula FN6191 돌연변이 삭제 지역과 후보 유전자 돌연변이 고기와 관련 된 식별 하 사용 되었다. M. truncatula FN6191 돌연변이, 빠른 중성자 사격 유도 삭제 돌연변이 컬렉션32 에서 원래 고립 된 토양 접종 후 하이퍼 nodulation 표현 형을 전시 하는 ( 테이블의 자료참조) 박테리아, 야생 유형 식물 달리 Sihorhizobium meliloti Sm1021

<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

돌연변이 유전자 기능 연구에 대 한 귀중 한 유전 자원 이다. 돌연변이 컬렉션을 생성 하려면 세 가지 유형의 mutagens 활용할 수 있습니다, T DNA 또는 transposon 등 생물, 에틸 methanesulfonate (EMS), 같은 화학 또는 이온화 방사선 같은 물리를 포함 하 여. 돌연변이 관찰의 유형에 돌연 사용에 따라 다릅니다. 이온화 방사선 유발 돌연변이, 돌연변이 또는 삭제, 복제, 또는 재배치 포함. T-DNA 또는 transposon-기반 mutagenesis는 변환에 취약 종으로 제한, 화학 또는 물리적 mutagenesis 종의 광범위 한 범위에 적용할 수 있습니다. 그러나, 전통적으로 화학 또는 물리적 mutagenesis에서 파생 하는 돌연변이의 특성 지도 기반 복제 방식을, 노동 집중과 시간이 소모 되는 사용 합니다. 자, 우리 감지 하 여 효율적으로 복사 번호 유사 (CNVs) 돌연변이에 빠른 중성자 사격 (FNB) mutagenesis에서 파생 된 특성 고밀도 게놈 배열 기반 비교 genomic 교 잡 (aCGH) 플랫폼을 적용할 수 있음을 보여합니다 개자리속 truncatula, 콩과 식물 종입니다. 전체 게놈 시퀀스 분석 50000 개 이상의 유전자 나 유전자 모델 M. truncatula에서 보여줍니다. M. truncatula 에서 현재, FNB 유발 돌연변이에서 150000 이상 m 1 라인을 대표 하는 게놈에 있는 유전자의 기능 연구에 대 한 귀중 한 유전 자원에서 파생 됩니다. 여기에 설명 된 aCGH 플랫폼 FNB 유발 돌연변이 M. truncatula에 특성화를 위한 효율적인 도구입니다.

그림 2 는 전체 게놈에서 돌연변이 WT 신호 대의 표준화 된 로그2 비율의 분포를 보여준다. CGH 데이터의 분석 공개 대략적인 전체 SUNN 유전자33 와 몇 가지 다른 염색체 4에 22 kb 삭제 주석 FN6191 돌연변이 (그림 2, 그림 3)에서 유전자. 후보자 삭제 된 지역 평균 정규화 된 로그<s...

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

이 프로토콜에 복사 번호 유사의 전체 게놈 배열 기반 비교 genomic 교 잡 (CGH) 분석 수행에 관심이 있는 연구자에 대 한 실험 단계 및 시 약, 장비, 및 분석 도구에 대 한 정보 제공 식물입니다.

어레이 기반 비교 Genomic 교 잡 플랫폼 효율적인 탐지의 복사 번호 유사 빠른 중성자 유도 개자리속 truncatula 돌연변이에 대 한

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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This workflow allows novice researchers to leverage advanced computational resources such as cloud computing to carry out pairwise comparative ...

Detection of Copy Number Alterations Using Single Cell Sequencing

단일 셀 시퀀싱을 사용하여 복사 번호 수선의 검출

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

여러 형광에 의한 간 염색체 안정 수차의 검출<em&gt; 현장에서</em&gt; 하이브리드 (mFISH) 및 방사선 조사 마우스의 스펙트럼 핵형 분석 (SKY)

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

빠른 실버 얼룩 프로토콜 사용 간단 하 고 효율적인 탐지의 SSR 마커는 비 변성 시키기 Polyacrylamide 젤을 사용 하 여

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

BIBAC-GW 이진 벡터를 사용 하 여 단일 복사본 삽입을 가진 유전자 변형 식물 생성

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

빠른 고 손쉬운 파이프라인 C. 선 충 을 사용 하 여 CRISPR/Cas9 Ribonucleoproteins에서 게놈 포인트 돌연변이 생성에 대 한

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 유전자 인간 말라리아 기생충의 조건부 돌연변이 게 P. falciparum 에 편집

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

제자리에 전체 산 교 잡에 의해 틱 용기에 Microbiota의 시각화

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...