1. 시료 채취 <ol><li> 적절한 동의와 개인의 혈액 샘플을 수집합니다. 각 개인에 대한, P100 튜브 (BD 진단, 프랭클린 호수, NY, USA)에 혈액의 4 ~ 5 ML을 그립니다. 비교를 위해 동시에 EDTA 튜브에 혈액을 수집합니다. </li><li> BD P100 관은 15.8 ML 스프레이 건조 K2EDTA 응고를 방지하고 혈장 단백질을 안정화하는 단백질 분해 효소 억제제의 동결 건조 독점 넓은 스펙트럼 칵테일이 포함되어 있습니다. 또한 기계적 분리기가 포함되어 있습니다. 처리가 발생할 때까지 전체 혈액을 수집 한 후, 하룻밤에 60 분 동안 실온에서 모두 튜브를 유지합니다. </li><li> 실온에서 15 분 동안 2천5백g에서 BD P100 튜브를 원심 분리기. 마이크로 원심 분리기 튜브에 기계적 분리 위에서 수집 된 플라즈마를 전송합니다. </li><li> -80에서 분리 아래 압축 혈액을 포함, P100 튜브를 저장 ° C가 DNA 내선을 시작할 준비가 될 때까지raction. </li></ol> P100 튜브의 혈액 (P100_DNA)에서 2.1 <ol><li> P100 튜브 -80에 저장됩니다 ° C 혈장 추출 후. 3~4개월에서 5 분 (그림 1A)를위한 물을 욕조에 37 °에서 냉동 및 해동에서 압축 된 혈액을 포함하는 P100 튜브를 검색 할 수 있습니다. 분리 위에 앉아 남아있는 혈장을 제거합니다. 구분 위의 P100 튜브 (그림 1B)의 - 17 % 자당 용액 (게시되지 않은 데이터 집에서 테스트 그라데이션 용액) 2 ㎖를 추가합니다. 2천5백그램에서 20 분 동안 실온에서 튜브를 원심 분리기,이 프로세스는 튜브의 상단에 기계적 분리기를 밀어 버피 코트 (그림 1C) 등 솔루션의 세 가지 레이어를 생성합니다. 수동 steriled 갈고리 금속 클립 (그림 1D)를 사용하여 구분을 추출합니다. </li><li> 각 튜브 (그림 1D)에서 기계적 분리기를 검색 한 후, 제거하고 discarD 최고 자당 층. 멸균 15 ML 원뿔 튜브에 버피 코트 (BC) 계층을 나타내는 중간 계층, 전송합니다. 3 ML에 버피 코트 볼륨을 높이려면 인산 버퍼 식염수 (PBS)를 추가합니다. 제조업체의 권장 사항에 따라 Qiagen의 Puregene 혈액 키트의 시약을 사용하여 버피 코트에서 DNA를 추출, 2,500그램의 원심 분리 속도 (대신 2천g의)를 제외하고. </li><li> 적혈구 (RBC)를 용해하고 제거하는, 버피 코트 3 ㎖에 RBC 용해의 10 ML을 추가합니다. 각 튜브를 여러 번 반전하고 배양하는 동안 가끔 반전 10 분 실온에서 알을 품다. 5 분 2,500 g에서 각 혼합물을 스핀 다운 및 뜨는 폐기하십시오. 15 초 동안 세포 용해 용액과 소용돌이 3 ㎖ 각 튜브에 남아있는 펠렛을 처리합니다. 1 단백질 침전 용액의 mL를 15 초 동안 소용돌이와 해물을 취급합니다. </li><li> 5 분 2,500 g로 원심 분리 포함 신선한 15 ML 원뿔 튜브에 뜨는을 전송100 % 이소프로판올 3 ㎖. 3 분 2,500 g에서 새로운 튜브 10 배와 원심 분리기를 반전. 뜨는을 취소하고 70 % 에탄올 1 ML을 추가합니다. 원뿔 관에게 DNA 펠렛을 이동시키다 씻어 몇 번 반전. 1 분 2,500 g에서 원심 분리기. 펠렛은 상온에서 3 분 (거꾸로 튜브를 배치)에 대한 건조 후 10 분, 미리 표시 1.7 ML의 모세관로 전송하여 37 ° C에서 수분 보충 용액 250 ml의 DNA를 일시 중단 할 수 있습니다. -80에서 튜브를 저장 ° C를 사용하기 전까지. </li></ol> EDTA Ttube의 피 (EDTA_DNA)에서 2.2 <ol><li> 일상 immunohematology 테스트를위한 전체 혈액 K 2 EDTA (에틸렌 다이아 민 테트라 초산)의 10.8 밀리그램 스프레이 코팅 및 -80 ° C.에 저장 플라스틱 vaccutainer 튜브에 수집 튜브는 기계적 분리기를 포함하지 않기 때문에, DNA 추출은 기계적 분리기 (2.1.1 및 2.1.2)를 포함하는 단계를 포함하지 않습니다. </li><li> 혈액 저장, DN의 3~4개월 이내이 물총새 플렉스 (써모 피셔 과학, 보스턴, MA, USA)를 사용하여 추출됩니다. 그것은 자기 입자 기술을 기반으로하고 세포 용해 / DNA 바인딩, 여러 가지 세척 단계와 DNA의 용출로 구성 자동화 DNA 추출 시스템입니다. </li><li> DNA 추출에 사용되는 장비는 물총새 플렉스 24 DNA 혈액 키트 (Qiagen의, 독일)입니다. </li></ol> 3. DNA 수량 및 품질 평가 게놈 DNA의 양, 품질, 무결성 picogreen, 분광학 및 전기를 포함 방법의 조합에 의해 평가되었다. <ol><li> 게놈 DNA의 농도는 Nanodrop 분광 및 picogreen 정량에 의해 결정됩니다. </li><li> 순도 Nanodrop 분광 광도계에 280분의 260 비율 측정에서 결정됩니다. </li><li> 샘플 (500 NG)도 DNA의 무결성 (저하)를 평가하기 위해 1 % 아가로 오스 겔에로드됩니다. </li></ol> 4. GenotypinG 분석 실험 및 품질 관리 <ol><li> 다섯 P100_DNA와 해당 EDTA_DNA 샘플은 무작위로 지정 Immunochip (면역 DNA 분석 BeadChip)를 사용하여 추가 분석을 위해 선택됩니다. Immunochip는 196,524 다형성을 포함하는 Infinium 유전형 칩과 immunogenetic 연구 5를 위해 디자인된다. </li><li> 각 샘플은 DNA 200 NG는, 조각난 증폭 시켰던하고 적절한 하이브리드 버퍼에 재현 탁하고 있습니다. </li><li> 변성 샘플은 16 시간의 최소 48 ° C에서 Immunochips에 교배되어 있습니다. </li><li> 하이브리드 후에 Immunochips는 단일 염기 확장 반응과 스테인드에 대해 처리됩니다. </li><li> immunochips은 다음 Illumina의 Hiscan 비드 어레이 리더 정규화 된 비드 강도 데이터는 Illumina의 GenomeStudio 소프트웨어에로드 유전자형으로 변환을 사용하여 몇 군데 있습니다. 유전자형은 GenomeStudio의 autocalling 알고리즘을 사용하여 호출합니다. 의 품질 관리콜금리 &lt;95 %의 단일 염기 다형성의 배제 (SNPs를)를 volves. </li><li> SNP 통화의 환율을 immunochip 분석 효율의 척도로 사용됩니다. 그것은 자동 유전자형 할당을 받기에 충분한 신호 강도 및 품질을 달성 칩 (196524 SNP를 각각의 칩에 인코딩)에 대한 분석의 총 수의 백분율로 계산됩니다. 고품질 DNA는 (260 / 1.8 280 비율 이상 및 열화의 유무) immunochip 분석에 포함 된 HapMap 제어 DNA로 적어도 동일한 통화 비율을 달성 할 것으로 예상된다. 품질 관리는 콜금리 &lt;95 % 각 데이터 세트와 SNP는 제외를 포함한다. </li></ol>

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관심 없음 충돌 선언하지 않습니다.

많은 인간의 질병은 유전 적 원인을 가지고 인간의 질병의 유전 기초를 탐구하는 것은 증가하고 고품질의 DNA를 요구한다. 유전자 연구에 사용 된 DNA의 가장 일반적인 소스는 전체 혈액 항응고제 있습니다. 대부분의 임상 실험실 표현형 결과 조사의 첫 단계로, 생화학 적 혈청 및 바이러스 검사를 실시하고 있기 때문에, 혈액은 종종 플라즈마 튜브에 수집됩니다. 플라즈마를 수집 한 후, 압축 된 혈...

<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

실험실 테스트는 혈액의 세포 또는 액체 부분에서 수행 할 수 있습니다. 다른 혈액 수집 관의 사용은 분석 할 수있는 혈액 (전혈, 혈장 또는 혈청)의 부분을 결정합니다. 인간 질환의 유전 적 기초를 공부에 관련된 연구소 게놈 / 유전자 분석을위한 DNA의 소스로 EDTA를 함유 vacutainer에서 수집 된 항 응고 전체 혈액에 의존하고 있습니다. 대부분의 임상 실험실 표현형 결과 조사의 첫 단계로 생화학 적, 혈청 학적 및 바이러스 검사를 실시하고 있기 때문에, 항 응고 혈액은 헤파린 함유 튜브 (플라즈마 튜브)에 수집됩니다. DNA와 플라즈마 게놈과 프로테옴 데이터 모두를 동시에 병렬 분석에 필요한 그러므로 때, EDTA (에틸렌 다이아 민 테트라 초산)과 헤파린 튜브 모두에​​서 혈액을 수집하는 것이 관례이다. 혈액은 하나의 튜브에 수집 혈장과 DNA 모두에 대한 소스 역할을 할 수 있다면, 그 방법은 기존의 마약을 발전 간주됩니다ODS. 혈장 추출 후 압축 혈액의 사용은 이렇게 처리 된 혈액 샘플의 양을 최소화하고 각 환자에서 필요한 샘플 수를 줄여, 게놈 DNA의 대체 소스를 나타냅니다. 이것은 궁극적 시간과 자원을 절약합니다. 혈장 단백질 보존 BD P100 혈액 수집 시스템은 인간의 혈액에서 더 나은 단백질 바이오 마커 발견 및 프로테오믹스 실험을 가능하게 혈액의 단백질 함량을 안정화하기 위해, 이전의 혈장 또는 혈청 수집 관 1 이상 개선 된 방법으로 만들어졌습니다. BD P100 관은 15.8 분무 건조 K2EDTA ㎖의 응고를 방지하고 혈장 단백질을 안정화하는 단백질 분해 효소 억제제의 동결 건조 독점 넓은 스펙트럼 칵테일이 포함되어 있습니다. 또한 원심 분리 후 혈장과 세포 펠렛 사이에 물리적 인 장벽을 제공하는 기계적 분리기를 포함한다. 몇 가지 방법이 응고 혈액 SA에서 DNA를 추출하기 위해 고안되었습니다mples 오래된 플라즈마 튜브에 2-4 모았습니다. 이 방법의 문제는 주로 튜브 (겔 구분) 내부 분리기의 종류와 관련된 응고 혈액을 복구하는 어려움, 단편화 또는 응고 분산의 불편과 분리 겔 응고 추출 방해 하였다. 우리는 새로운 BD P100 튜브에 그려진 혈액에서 게놈 DNA를 추출하고 정화 첫 번째 방법을 제시한다. 우리는 P100 튜브에서 EDTA 튜브에서 그에게 DNA 샘플의 품질을 비교합니다. 우리의 접근 방식은 간단하고 효율적입니다. ) 혈액 수집, 2 기계적 분리기와 자당의 조합을 사용하여 기계적 분리기의 제거 및을 1) 플라즈마 BD P100 (BD 진단, 스파크, MD, USA)의 사용이 관 : 그것은 다음과 같이 네 가지 주요 단계를 포함 멸균 클립 금속 걸이, 3) 백혈구, 4를 포함 버피 코트 층의 분리)에서 게놈 DNA의 분리일반 상업 DNA 추출 키트 또는 유사한 표준 프로토콜을 사용하는 버피 코트.

DNA의 품질 평가 및 농도 측정 P100_DNAs의 수율 EDTA_DNAs (표 1과 2)에 비해 유의하게 낮았다. 그 280분의 260 비율 값으로 표시 DNA 순도 P100_DNA 및 EDTA_DNA 샘플 세트 (테이블 1과 2) 모두 비슷합니다. DNA의 품질은 샘플의 각 집합 일부 저하가 EDTA_DNA 샘플에서 볼 수 있지만, 같은 (그림 2) 빛 번짐으로 표시 내에서 비교적 균일 한 것입니다. 분해의 더 많...

Watch this Scientific Journal Video about A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation at JoVE.com

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

우리는 플라즈마 / 혈청 수집 전혈로부터 게놈 DNA를 분리하는 새로운 방법을 설명합니다. 혈장 수집 후, 압축 된 혈액은 일반적으로 삭제됩니다. 우리의 새로운 방법은 기존의 방법에 비해 상당한 개선을 대표하고 추가 혈액을 요청하지 않고, DNA와 단일 컬렉션에서 사용할 수있는 플라즈마를 만든다.

플라즈마 단백질 보존을위한 혈액 수집 장비로부터 게놈 DNA를 추출하는 실제적이고 새로운 방법

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 ...

a-practical-novel-method-to-extract-genomic-dna-from-blood-collection

Research

JoVE Journal

Biology

41.9K 조회수.  Emory University School of Medicine and Children's Health Care of Atlanta. 우리는 플라즈마 / 혈청 수집 전혈로부터 게놈 DNA를 분리하는 새로운 방법을 설명합니다. 혈장 수집 후, 압축 된 혈액은 일반적으로 삭제됩니다. 우리의 새로운 방법은 기존의 방법에 비해 상당한 개선을 대표하고 추가 혈액을 요청하지 않고, DNA와 단일 컬렉션에서 사용할 수있는 플라즈마를 만든다.

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

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

연구

생물학

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.

미국 호르세쇼 게, Limulus Polyphemus의 혈액 수집

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.

소일스 및 세디먼츠에서 높은 분자량의 게놈의 DNA의 추출

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.

자기 활성 셀 정렬 (LIMACS)를 사용하여 지질 소포 - 중재 동질 크로마 토그래피 : 단백질 지질 상호 작용을 분석하는 소설 방법

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.

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 ...

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 ...

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;

비전문가를위한 계통 발생에 대한 실용 가이드

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.

키나제 억제제에 대해 내성 돌연변이의 검사 및 검증을위한 방법

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.

성인 Zebrafish의에서 혈액 검사에 대한 반복 채혈

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.

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

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