The authors have no acknowledgements.

1. 라벨 반응 <ol><li> 사용할 준비가 -20 C에서 접시와 상점 96 웰으로 제조업체가 권장하는 농도에서, 미리 나누어지는 메틴 3, 5 표지 dUTPs, 레이블이없는 뉴클레오티드 랜덤 프라이머를 사용하기 전에. 이 프로토콜, 분취 적절한 표지 된 dUTP의 10.5 μL 10.5 μL 뉴클레오티드와 표지되지 않은 각 웰 랜덤 프라이머의 목적. </li><li> 빛으로부터 보호는 약 1 시간 동안 12 ° C에서 뉴클레오티드 프라이머가 즉시 사용 96 웰 플레이트를 해동. </li><li> 해동 후, 빛으로부터 보호 적어도 30 분 동안 실온에서 평형이 플레이트. </li><li> 적어도 15 분 동안 60 ° C에서 DNA 샘플을 평형. </li><li> 액체 핸들링 로봇을 이용하여 96 웰 플레이트의 각 웰에 클레아없는 물을 분배. 주 : 물의 부피는 각각의 입력 샘플 DNA의 농도에 의존 할 것이다 NG 50 / UL의 최종 농도 야기하기에 충분해야한다. </li><li> 액체 한 사용dling 로봇, 96- 웰 플레이트의 웰에 피펫 1 μg의 DNA가 (1.5 참조) 20 μL에 전량을 가지고. 주 : 결과 데이터의 품질이 저하 될 수 있지만, DNA의 소량이 사용될 수있다 (귀중한 샘플 400 NG 아래로 입력 DNA 량을 사용할 수있다). </li><li> 액체 핸들링 로봇을 이용하여 DNA의 희석 플레이트의 각 웰에 평형화 뉴클레오티드 프라이머의 20 μl를 옮긴다. </li><li> 스트립 캡 (단단히 밀착이 중요하다)와 함께 96 웰 플레이트를 밀봉합니다. </li><li> 가열 뚜껑 PCR 기계에서 10 분 동안 99 ° C에서 DNA를 변성하고 어닐링 프라이머 5 분 동안 얼음 냉각 스냅. </li><li> 액체 핸들링 로봇을 이용하여 각 DNA를 철저히 혼합하고 피펫으로 엑소 클레 나우 DNA 폴리머 라제 효소의 10 μL를 추가한다. 참고 : 전체 볼륨이 50 μl를해야한다. </li><li> 96 웰 플레이트를 밀봉하고 16 시간 동안 37 ° C에서 품어. 참고 : 4 시간의 짧은 배양 그러나의 O / N 충분하다cubation 더 편리 할 수​​ 있습니다. </li><li> 액체 처리 로봇을 사용하여, 각 웰에 정지 버퍼의 5 μl를 추가합니다. 주 : 라벨에 사용되는 반응 시약의 개요를 표 1에 나타낸다. </li></ol> 비법 인 뉴클레오티드 2. 제거 <ol><li> 실리카 막 기반 스핀 열 및 전용 스핀 열 처리 로봇 (스핀 열을 수동으로 처리 될 수있다)를 사용하여 비법 인 뉴클레오티드를 제거합니다. 제조업체의 지침을 따르십시오. <ol><li> 웰 2 ㎖의 튜브에 각각의 내용을 전송; 잘 ID 년대와 사전 라벨이는. 주 :이 단계는 액체 핸들링 로봇을 이용하여 수동으로 또는 바람직하게 수행 될 수있다. </li><li> 스핀 컬럼 처리 로봇을 사용하면, 2 ㎖의 튜브, DNA 정제 스핀 컬럼과 연관된 버퍼와로드 로봇. </li><li> 실리카에 표시된 DNA에 결합하는 라벨링 반응 250 ㎕의 높은 소금, DNA 결합 버퍼 (완충 PB)를 추가합니다막. </li><li> 500 μL 세척 버퍼 (PE 버퍼)로 두 번 막을 세척하여 불​​순물을 제거합니다. </li><li> ~ 12 μL의 부피 정제, 표지 된 DNA를 회수하는 막에 15 μL 저염 용출 완충액 (EB 버퍼)에 추가. </li></ol></li></ol> 3. 그리고, Hyb 쌍을 결합 <ol><li> 시아닌 3, 5 표지 DNA의 적절한 쌍을 결합, COT-1 DNA, 제조업체에서 제공 차단 믹스와 액체 처리 로봇을 사용하여 제조업체에서 제공 하이브리드 버퍼입니다. 참고 : 공유 CNVs는 문제가되지 않습니다 경우 이러한 HYB 쌍 (예를 들어, 우리가 일상적으로 그들은 매우만큼, 같은 두개골 조기 유합 환자 대 신 이형성증 환자 등의 표현형 - 일치하지 않는 샘플을, 쌍 샘플 대 샘플과 기준, 또는 샘플이 될 수 있습니다 ) 같은 임상 적으로 유의 CNV 운반 가능성. 기준 DNA를 사용하는 경우, 상용 전원 DNA를 권장하고, 시료와 함께 처리되어야한다. <ol><li> 액체 취급 RO 사용로봇, COT-1 DNA (3333 NG / μL)을 추가 제조업체 제공 각 웰 1.1 μL의 COT-1 DNA, 4.95 ㎕의 블로킹 믹스 24.75 포함되도록 96 웰 플레이트의 각 웰에 혼합하고 혼성화 블로킹 완충액 μL 하이브리드 버퍼입니다. </li><li> 각 웰에 해당 시아닌 3 표지 DNA의 9.35 μl를 추가합니다. 사전 젖은 각각의 팁은 정확성을 피펫 팅 향상. </li><li> 각 웰에 해당 시아닌 5 표지 DNA의 9.35 μl를 추가합니다. 사전 젖은 각각의 팁은 정확성을 피펫 팅 향상. </li><li> 1 분 동안 96 웰 플레이트와 소용돌이 인감. 각 웰의 바닥에 내용을 수집하기 위해 96- 웰 플레이트 스핀. </li></ol></li></ol> 4. 하이브리드 <ol><li> 65oC에 하이브리드 오븐을 예열 한 백업 슬라이드 및 각 배열 슬라이드 한 하이브리드 챔버를 prewarm. <ol><li> 배열에 적용되기 전에 미리 하이브리드 배양에 표시된 DNA를 변성. 24 시간 동안 회전 오븐에서 하이브리드를 수행합니다. </li><li> 30 분 동안 70 ° C에서 96 웰 플레이트를 인큐베이션 후 적어도 5 분 동안 37 ° C에서 떠난다. </li><li> 42 ° C에서 가열 된 플랫폼에서 작업 사용하기 전에 하이브리드 실에 각각의 백업 슬라이드를로드합니다. 가스켓 슬라이드의 투명한 부분은 하이브리드 챔버의 &#39;윈도 윙&#39;부분과 정렬되었는지 확인합니다. </li><li> 피펫 이전에 제조 된 하이브리드 믹스의 42 μl의 슬라이드를 백업 어레이의 적절한 위치에 (섹션 3 참조). 사전 젖은 각각의 팁은 정확성을 피펫 팅 향상. 피펫 천천히 확인하고 각 위치의 중심에 액상 고무 링 경계를 만지지 않습니다. </li><li> 백업 슬라이드에 모든 위치가 작성되면,주의 깊게에 배열 슬라이드를 낮추고 하이브리드 챔버를 조립한다. 그것을 쓰기로 배열의 측면 등판이 슬라이드를 향하고 있는지 확인하십시오. 완전히 하이브리드 실 나사를 조이십시오. </li><li> 조립 된 하이브리드 챔버를 검사합니다. E이 고무 링 경계 외부 혼성화 믹스의 누설이없고, 혼성화 챔버의 수직 단부에 얹혀 때마다 기포 높이 ~ 4mm로되어 nsure. 하이브리드 챔버를 돌려 위치 및 이동되지 붙어있는 기포가 없는지 확인합니다. 이들 중 하나라도있을 경우, 챔버를 벤치에 예리한 탭을 준다. 모든 기포가 이동하고 있는지 확인하십시오. </li><li> 24 시간 동안 65 ° C에서 회전 오븐에 하이브리드 챔버를 놓습니다. 오븐 로터가 균형을 확인하십시오; 필요한 경우 다른 빈 챔버를 사용합니다. </li></ol></li></ol> 5. 세탁 스캔 슬라이드 <ol><li> 초과 표지 DNA를 제거한 다음 각 프로브의 형광을 측정하기 위해 스캔 배열을 씻으십시오. <ol><li> 오븐과 분해에서 하이브리드 챔버를 가져 가라. 배열 및 가스켓 슬라이드 붙어됩니다; 세척 버퍼 1이 잠수함과 간격을 캐 내려고 플라스틱, 평면 깨끗 포셉 한 쌍을 사용합니다. </li><li> 디가스켓 슬라이드를 scard 버퍼 1에 잠긴 랙에 배열 슬라이드를 배치합니다. </li><li> 격렬하게 교반, 5 분 동안 신선한 세척 버퍼 1 ~ 700 ml의에 배열 슬라이드를 씻으 (벼룩과 자석 교반기를 사용). </li><li> ~ 700 ml의 세척 버퍼 (2)에 배열 슬라이드를 전송하고 격렬하게 교반, 90 초 동안 세척한다. 부드럽게 세척 버퍼 2 중 배열 슬라이드를 올려, 그들은 건조 등장한다. </li><li> 슬라이드 방지기를 사용하여 스캐너의 슬라이드 홀더에 배열 슬라이드를 넣습니다. 배열 스캐너로 슬라이드를 넣고 배열 ​​제조업체의 지침에 따라 검사합니다. </li></ol></li></ol>

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The authors have nothing to disclose.

어레이 CGH는 triploidy로 균형 재 배열 또는 배수성 이상을 감지하지 않습니다. 또한, 낮은 수준의 모자이크 불균형을 감지 할 수없는 문제점이있다. 그러나, 배열 CGH는 많은 세포 유전학 실험실에서 대체하고 G 줄무늬 염색체 분석보다 CNV 검출을위한 높은 해상도를 가지고있다. 따라서 게놈 전체의 CNV 감지를위한 황금의 현재 표준입니다. 이것은 향후 차세대 시퀀싱 기술로 대체 될 수 있지만, 현재,...

이것은 염색체 결실 또는 세그먼트들의 추가 복사본들이 지적 장애, 및 dysmorphism congentical 기형을 일으킬 그 수년 동안 알려져 있고, 어떤 경우에는 하나의 유전 적 증후군을 야기하고있다. 그러나 이러한 변화의 게놈 전체의 검출을위한 2000 년대 중반까지 사용할 수있는 유일한 기술은 할 수없는 불균형의 지역 및 특성에 따라 5-10Mb 주위의 해상도를 가지고 염색체 분석을, 줄무늬 G, 그리고 어떤 재료가 동일한 밴딩 패턴 다른 염색체 영역에 의해 대체 된 염색체 이상을 검출한다. 이러한 현장 하이브리드 멀티 플렉스 결찰 특정 프로브 증폭 형광 등의 보조 세포 유전 학적 기술이 의심되는 특정 syndromic 불균형의 경우, 특정 유전자좌의 심문에 사용할 수 있었지만,이 배열 비교 게놈 교잡의 도입까지이지 않았다 (배열 CGH) 일상적인 임상 진단의에카피 수의 게놈 넓은 감지 (CNVs) 변종 서 비 스 2-5 (일반적으로 1백20킬로바이트 정도) 크게 증가 해상도로 가능하게되었다. 일부 지역은 일반 인구의 6-7에 널리 퍼져에 대한 조사 연구와 함께 임상 서비스 작업은 다른 CNVs 반면, 이전에 발견 할 수없는, 자폐증과 간질 8-11로 neurodisabilities과 관련된, CNVs 것으로 나타났습니다. 이 문서에 설명 된 프로토콜은 우리의 영국 국민 건강 서비스 (NHS) 임상 진단 실험실에서 사용된다 우리는이 국가 재정 지원 서비스 비용을 최소화하기 위해 새로운 하이브리드 전략, 배치 테스트 및 로봇을 사용합니다. 아래 설명 프로토콜 이전에, 고품질의 DNA가 적절한 출발 물질, 일반적으로 혈액, 세포 배양 또는 조직 샘플로부터 추출한다. 280 흡광도 비 (B한다 :이 측정법 농도를 측정하는데 사용될 수 있고 (260)을 확인 (&gt; 50 NG / UL이 있어야한다)전자 1.8 ~ 2.0). 젤 전기 영동은 DNA가 크게 저하없이 높은 분자량 있는지 확인하는 데 사용할 수 있습니다. 이 프로토콜은 자동화 된 액체 처리 로봇을 사용하여 런타임 당 96 샘플에 레이블 높은 처리량 실험실을 위해 설계되었습니다. 그러나, 낮은 처리량없이 자동화 실험실 위해 적응 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CGH microarray 8 x 60K</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">G4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH wash buffers</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH hybridisation buffer</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">5188-5380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Minelute purification kit</td>
			<td style="width:71px;">Qiagen</td>
			<td align="right" style="width:119px;">28006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Array CGH labelling kit</td>
			<td style="width:71px;">Enzo</td>
			<td style="width:119px;">ENZ-42672-0000</td>
			<td></td>
		</tr>
	</tbody>
</table>

array comparative genomic hybridization (array cgh) for detection of genomic copy number variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

혼성화 된 각 프로브 어레이에 적색, 녹색의 형광체의 혼합물로서 가시화된다 (도 1 참조). 각 프로브에 대한 녹색 형광 신호에 빨간색의 비율은 스캐너에 의해 정량화하고 관련 소프트웨어는 자신의 게놈 위치에 따라 LOG2 비율 이러한 플롯, 외부 프리셋 경계 떨어지는 영역을 식별합니다. 결과 배열 트레이스는 genomically 불균형으로 식별 영역의 해석을 할 수 있습니다. 예를 들어, 윌?...

Watch this Scientific Journal Video about Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants at JoVE.com

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

유전자 복제 수 변이의 검출을위한 어레이 CGH는 G-밴드 핵형 분석을 대체하고있다. 이 문서는 기술 및 진단 서비스 실험실에서의 응용 프로그램을 설명합니다.

게놈 복사 번호 변종의 탐지를위한 배열 비교 게놈 교잡 (배열 CGH)

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

array-comparative-genomic-hybridization-array-cgh-for-detection

Research

JoVE Journal

Biology

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

19.4K 조회수.  Guy's & St Thomas' NHS Foundation Trust.  유전자 복제 수 변이의 검출을위한 어레이 CGH는 G-밴드 핵형 분석을 대체하고있다. 이 문서는 기술 및 진단 서비스 실험실에서의 응용 프로그램을 설명합니다. 

비디오: Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

연구

생물학

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

전체 게놈 배열 비교 게놈 하이브 리다이 제이션의 기술의 데모

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. Lightweight recording devices carrying a high density array of tetrodes allow for the simultaneous monitoring of the activity of tens to hundreds of individual neurons. Here we describe a protocol for the fabrication of a micro-drive array with twenty one independently movable micro-drives. This device has been used successfully to record from hippocampal and cortical neurons in our lab. We show how to prepare a custom designed, 3-D printed plastic base that will hold the micro-drives. We demonstrate how to construct the individual micro-drives and how to assemble the complete micro-drive array. Further preparation of the drive array for surgical implantation, such as the fabrication of tetrodes, loading of tetrodes into the drive array and gold-plating, is covered in a subsequent video article.

Micro-drive Array for Chronic in vivo Recording: Drive Fabrication

In this protocol we demonstrate how to fabricate a micro-drive array for chronic electrophysiological recordings in rats.

만성에 대한 마이크로 드라이브 배열 생체내에 녹화 : 드라이브 제작

Chronic recording of large populations of neurons is a valuable technique for studying the function of neuronal circuits in awake behaving rats. ...

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.

대형 삽입 환경 게놈 라이브러리 제작

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

의 DNA Microarrays : 샘플 품질 제어, 배열 하이브 리다이 제이션 및 스캔

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

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.

게놈 MRI - 게놈 DNA 시퀀스 내의 패턴을 학습에 대한 공개 자료

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.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

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.

파편을 사용하여 풀링된 장면에서 드문 게놈 변종의 탐지

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryote의 게놈 변환<em&gt; Ostreococcus 타우 리</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

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.

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

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 ...