This protocol development was supported by the Bill and Melinda Gates Foundation Grand Challenges in Global Health grant 37774. Real-time PCR reagents and advice were provided by Abbott Molecular Inc. Des Plaines, IL. 8E5-LAV cells were provided by the Virology Quality Assurance Laboratory, Rush Presbyterian; St. Luke's Medical Center. HIV-1 negative blood was provided by Core Lab, NorthShore University HealthSystems, Evanston, IL. Thanks to Mark Fisher for photography help.

윤리 문 : 본 연구에 사용 된 전체 혈액 표본은 인체를 이용한 연구로 간주되지 않습니다. 시편은 만족 임상 진단 목적을 위해 수득하고,이 시험편의 나머지 부분은 FINA 연구 시험에 제공 하였다. 시편은 연구자 용이 개인의 신원을 확인할 수 없었다되도록 부호화 하였다. FINA 샘플 준비 모듈 1. 준비 <ol><li> A A 2.6 mm의 35 X 35 mm 2 두께의 100 % 코튼 패드를 절단하여 패드를 모래 바닥 준비합니다. 표본 당 하나의 패드를 잘라. </li><li> 다음 망치 중심 펀치 구멍을 사용하여 테이프의 중앙에 7.14 mm 직경의 구멍을 펀치 (50 () w mm 25 mm (H)) 파라핀 필름의 절단 조각이 용지 양면 테이프 플라스틱 파라핀 필름을 준비한다. 표본 당 하나의 테이프를 준비합니다. </li><li> 8.35 mm 직경의 원형 펀칭에 의해 결합 된 유리 혈액 분리막 (캡처 막) 디스크를 준비망치 중심 구멍 펀치를 사용하여. 표본 당 하나의 디스크를 펀치. 디스크는 2.3 ㎛의 입자 보유 능력을 갖는다. </li><li> 겸자를 이용하여 블롯 패드의 중앙에 포착 디스크를 배치하여 FINA 샘플 준비 모듈을 조립한다. 파라핀 테이프의 구멍이 노출 된 캡처 디스크의 중심 잎되도록 캡처 디스크 위에 파라핀 필름 테이프를 배치했다. </li><li> 블로 팅 패드를 충당하기 위해 약간 파라핀 테이프를 스트레칭하고 디스크의 모든 가장자리 주위에 블로 팅 패드에 붙어 단단히 파라핀 테이프를 눌러 디스크와 블로 팅 패드 사이의 최대 접촉을 확인합니다. </li><li> 나중에 사용하기 위해 실험 또는 비축에 필요한만큼 모듈을 확인합니다. </li></ol> qPCR의 튜브 (2)의 제조 <ol><li> 양면 polye의 원형 펀칭하여 형광 검출을 차단하는 멤브레인을 방지 qPCR에 튜브 측으로 세포 포획 막을 고정하는 5.1 mm 테이프 디스크를 준비테이프의 시트에서 망치 구동 펀치 진단 테이프를 오스터. 표본 당 하나의 테이프 디스크를 준비합니다. </li><li> 테이프의 스티커 라이너의 한면을 제거합니다. 한쪽에 튜브의 바닥쪽으로 튀어 나와, 200 μl의 PCR 튜브에 테이프를 넣습니다. 두 번째 스티커 라이너를 제거합니다. 튜브는 이제 디스크 제조를 수신 할 준비가되어있다. </li><li> 나중에 사용하기 위해 실험 또는 비축에 필요한만큼의 튜브를 생성합니다. </li></ol> 3. 고안 혈액 표본 참고 : 정품 시험편을 준비하는 경우, 4 단계로 진행합니다. <ol><li> 바이러스학 품질 보증 실험실에서 얻은 HIV-1 프로 바이러스의 단일 복사본을 품고 해동 8e5 - LAV 셀 (15) (VQA, 러쉬 장로교 / 세인트 루크 메디컬 센터, 시카고, IL.). 참고 : 그들은 / μL 4,000 세포의 농도로 동결 세포 펠릿으로 저장됩니다. 이전 9 바와 같이 세포 수를 확인 하였다. </li><li> 산이 준비/ μL 일 400 세포의 농도 범위로 배지 (90 % 소 태아 혈청, 10 % 디메틸 술폭 시드)를 동결 IAL 희석. </li><li> 미세 원심 튜브에 희석액에서 각각 10 μL 피펫 셀. 8e5 - LAV 복사 번호 10-4,000의 표준 곡선을 만들기 위해 각각의 튜브에 신선한 HIV-1 네거티브 EDTA 처리 된 전체 혈액 샘플 100 μl를 추가합니다. 부드럽게 튜브의 5 배를 바닥을 쓸어 넘겨 섞는다. </li></ol> 4. FINA 추출을 수행 <ol><li> 110 μL 전혈 단계 3.3에서 세포)에 1 % (11 ㎕의 10 % 트리톤 X100 최종 농도 트리톤 X100)을 첨가하여 다운 Lyse 혈구. </li><li> 부드럽게 튜브의 5 배를 바닥을 쓸어 넘겨 섞는다. 혈액은 반투명 빨간색을 설정해야합니다. </li><li> 피펫 캡처 디스크에 혈액 표본의 모든. 참고 : 파라핀 테이프 캡처 막 사이의 물이 꽉 밀봉 피가 막 흐르는 것을 보장하고, 캡처 m 사이 좋은 접촉embrane 및 블로 팅 패드 어셈블리는 빠른 샘플 위킹을 보장합니다. </li><li> 샘플 필터를 통해 흡수 할 수 있습니다. 주 : 모세관 현상에 블롯 패드에 피 파쇄 심지를 캡처 디스크 표면의 게놈 DNA를 캡쳐. 이 매트를 표시 할 때 해물 디스크에 배어있다. </li><li> 캡처 디스크에 600~1,000 ㎕의 10 mM의 NaOH를 드롭 현명한을 추가합니다. 주 : 세척 완충액은 600 μL의 대량 첨가로 첨가 할 수있다. 버퍼는 소수성 파라핀 테이프에 액 적을 형성하고, 약 20 초간의 디스크로 구멍을 통해 심지 할 것이다. 이 필터는 헤모글로빈의 흰색을 나타내는 통관에 빨간색으로 변경됩니다. </li><li> 집게와 블로 팅 패드에서 디스크를 분리하고 준비된 PCR 튜브에 테이프에 디스크를 적용합니다. 필터에 남아있는 붉은 색이있는 경우 튜브에 배치하기 전에 필터를 취소 세척 버퍼의 50 ~ 100 μl를 추가합니다. 참고 : 가끔, 초과 압력이 준비 중에 적용블로 팅 패드에서 분리 동안 멤브레인 층의 파티션에서 FINA는 모듈의 결과. 이러한 디스크를 폐기하고 새로운 샘플을 준비합니다. </li><li> 필터는 PCR 튜브에 배치 한 후, 바로 샘플을 분석 할 수 있습니다. 또한, 상자 포함 된 황산 칼슘 건조 건조 하룻밤은 다음 분석을위한 준비가 될 때까지 실리카 건조 및 저장과 호일 주머니에 모자 장소. </li></ol> 5. qPCR에 반응 <ol><li> 이전에 9,11 바와 같이 HIV는 특정 프라이머 및 프로브를 사용하여 반응 당 100 μL qPCR에 마스터 믹스를 준비합니다. 내부 증폭 제어 증폭 억제제의 존재를 모니터링하고 음의 샘플이 진정한 부정적 있는지 확인을 포함합니다. 필터가 완전히 마스터 믹스 필터 반응을 통해 튜브의 측면에 고정 된 상태를 유지하는 것이 포함되어 있는지 확인합니다. </li><li> 이전 9 바와 같이 실시간 PCR 기기를 사용하여 증폭을 수행한다. </li></ol>

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The authors have nothing to disclose.

빠른 치료 액세스 링크 EID 의한 HIV 감염 16 유아 사망률 감소하는 것으로 입증되었다. 때문에 유아의 혈액 모체 항체의 지속성 신속 HIV 항체 테스트는 HIV에 노출 된 유아의 상태를 결정하는 유틸리티를 제한하고있다. 보건기구 (WHO)는 HIV-1 양성 산모에게서 태어난 모든 유아는 바이러스 학적 검사 (17)를 사용하여, 연령 4 ~ 6 주에 테스트해야하는 것이 좋습니다. 우리는 (61) 남아프리?...

여러 보고서를 모두 사용할 수있는 절묘한 감성과 분자 진단의 특이성을 목적으로 현장 진료 (POC) 장치 1-5 사용하기 위해 종이 - 또는 멤브레인 기반 추출 방법의 개발을 논의했다. 세계 보건기구 (WHO) 성병 진단 이니셔티브는 POC의 (경제적, 민감한, 특정, 사용자 친화적 인, 신속하고 강력한 장비가없는 그것을 필요로하는 사람들 배달)을 설명하는 이상적인 특성 안심 용어를 만들어 낸 시험 6. 이 가이드 라인 중 장비가없는 특징은 분자 진단 달성하기 위해 특히 도전이다. 그러나, 필드에있는 모든 혁신은 가장 필요로하는 사람들을 도달 목표를 진행하며, 희망은 기존의 기술 (7)을 적용하여 테스트 성능을 단기적 개선이 있습니다. 여기에서 우리는 전체 혈액에서 DNA를 추출하는 간단한 프로토콜을 설명그 복잡한 화학 또는 실험실 장비를 필요로하지 않습니다. FINA (핵산 여과 분리) 시료의 제조 방법은 원래 샘플에 응답 케어 포인트 (POC)의 일부 정량적 PCR 같이 HIV-1 프로 바이러스를 검출하기 위해 전혈로부터 백혈구 DNA를 추출하기 위해 개발 된 제한된 리소스 설정 8-11에 사용 (qPCR에) 초기 유아 진단 (EID) 플랫폼입니다. FINA 추출 가역적 카오 트로픽 제 (12)의 존재 하에서 DNA 결합 실리카 막, 실리카 코팅 된 상자성 입자를 사용하는 종래의 정제 방법과 다르다. 대신, FINA 직접 전체 혈액에서 세포의 DNA를 추출하기 위해 분리막을 통해 수직 여과를 사용합니다. 혼입 된 DNA를 함유하는 멤브레인은 PCR 튜브에 직접 배치 및 하나 이후 즉시 증폭 9 건조 PCR 반응 또는 공기를 사용할 수있다. 어떤 카오 트로픽 제, 페놀 또는 알코올이 샘플의 추출에 이용되지 않으며, elimina팅 추출 처리 (13, 14)로부터 유도 qPCR의 강력한 억제제를 제거하기 위해 필요한 광범위한 세척 단계. FINA 막을 시험편 멤브레인에 추가되기 전에 세포 용해 (11)에 의해 해방 된 세포 중 9 또는 게놈 DNA를 포착 할 수있다. 셀 캡처, 전체 피가 막에 직접 추가됩니다. 세포를이어서 10 mM의 수산화 나트륨을 첨가함으로써 막에 용해된다. 이 방법의 장점은 단지 3 단계를 포함하는 것이있다 : 1) 샘플을 첨가하는 단계; qPCR에 관 2) 세포 용해 / 세척 및 3) 필터 디스크 위치. 이 방법의 단점은 막 만의 막 디스크의 직경에 비례 셀들의 정의 된 번호를 저장할 수 있다는 것이다. EID 필요한 검출 한계에 도달하기 전혈 100 μL은 qPCR에 튜브를 배치하기에 너무 큰 필터를 수반 샘플 입력을 요구한다. 컬렉션에 추가하기 전에 샘플을 세제로 혈액 세포를 용균막을 처리하는 단계를 추가하지만 동일한 샘플 크기보다 작은 필터를 사용할 수있다. 우리는 높은 재현성, 단일 복사본 검출하고,이 테스트 구성 (11)를 사용하여 혈액 100 ㎕에서 HIV-1 프로 바이러스의 DNA의 한 작은 10 복사본의 정량을 입증 할 수 있었다. 이 보고서에서 우리는 원래 실험실 사용하기 위해 개발로 FINA 프로토콜을 설명합니다. FINA 시료 전처리 모듈로 알려진 막 / 필터 샌드위치 큰 일괄 적으로 준비하고 나중에 사용하기 위해 비축 할 수있다. 표본 경우이 과정이 2 분 소요 크기의 배치를 변화에서 수행 할 수 있습니다 추​​출 할 수 있습니다. qPCR에 바로 실행될 수 있거나 qPCR을 수행하는 것이 편리 할 때까지 삽입 된 DNA를 함유하는 필터를 저장할 수있다. 이 방법은 낮은과 높은 리소스 설정 모두에서 시료의 일상적인 분석을위한 매우 편리합니다.

<table border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:432px;">Fusion 5</td>
			<td style="width:179px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">8151-9915</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">707 blotting pad</td>
			<td>VWR International</td>
			<td>28298-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PARAFILM M</td>
			<td>VWR International</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3M Double-Coated Polyester Diagnostic Tape&#160;</td>
			<td>3M Medical Specialties</td>
			<td>9965</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#181;l qPCR strip tubes</td>
			<td>Agilent</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">optical strip caps</td>
			<td>Agilent</td>
			<td>401425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mx3005p qPCR System</td>
			<td>Agilent</td>
			<td>401456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td>A.C.S. Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>BioXtra</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>for molecular biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum, certified, U.S. origin</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 3/16&#34; hole diameter (5.1 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 1/4&#34; hole diameter (7.14 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hammer driven small hole punch 5/16&#34; hole diameter (8.35 mm)</td>
			<td>McMaster Carr</td>
			<td>3424A23</td>
			<td></td>
		</tr>
	</tbody>
</table>

filtration isolation of nucleic acids:  a simple and rapid dna extraction method

FINA 핵산 여과 분리 미만에서 2 분에 전혈 세포에서 DNA를 추출 분리막 및 흡수 패드를 통해 여과에 수직을 이용하는 신규 추출법이다. 혈액 시료는 세제 혼합 간단히 처리 및 분리막을 피펫에 의해 적용된다. 해물이 분리막의 표면에, 게놈 DNA를 캡처, 모세관 현상에 의한 블롯 패드에 심지. 추출 된 DNA는 PCR 억제제 흡수 블롯 패드에 악 상기 간단한 세척 단계 동안 막에 유지된다. 혼입 된 DNA를 함유하는 멤브레인은 추가의 정제없이 PCR 반응에 첨가된다. 이러한 간단한 방법은 실험 장비를 필요로하지 않고 쉽게 저렴 실험실 용품으로 구현 될 수있다. 여기에서 우리는 초기의 모델로 100 μL 전체 혈액에서 HIV-1 프로 바이러스의 DNA의 고감도 검출 및 정량을위한 프로토콜을 설명쉽게 다른 유전 적 목표에 적용 할 수있는 HIV의 영아 진단.

8e5 - LAV 세포 아군 전체 혈액에서 프로 바이러스 DNA를 추출하기위한 워크 플로는 그림 1과 같다. 2는 FINA 샘플 모듈을 보여주고 qPCR에 튜브를 준비 그림. 인위적인 시험편 (도 3)의 표준 곡선에 도시 된 바와 같이,이 방법은 다른 사본 번호에 8e5-LAV 세포에서 HIV-1 프로 바이러스의 효율적인 증폭을 허용한다. PCR 효율 = -1 + 10 <s...

Watch this Scientific Journal Video about Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method at JoVE.com

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

우리는 여기에서 정량적 PCR에 의해 검출 된 전혈로부터 HIV 프로 바이러스 DNA의 단순하고 신속한 종이 기반의 DNA 추출 방법을 설명한다. 이 프로토콜의 다른 유전자 마커의 검출 또는 다른 증폭 방법을 이용하여 사용하기 위해 확장 될 수있다.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

filtration-isolation-nucleic-acids-simple-rapid-dna-extraction

Research

JoVE Journal

Biology

18.0K 조회수.  Northwestern University.  우리는 여기에서 정량적 PCR에 의해 검출 된 전혈로부터 HIV 프로 바이러스 DNA의 단순하고 신속한 종이 기반의 DNA 추출 방법을 설명한다. 이 프로토콜의 다른 유전자 마커의 검출 또는 다른 증폭 방법을 이용하여 사용하기 위해 확장 될 수있다. 

비디오: Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

핵산의 NanoDrop Microvolume의 Quantitation

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

연구

생물학

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

마이크로 볼륨 바이오 사양 나노 분광 광도계를 사용하여 핵산 및 단백질 농도 결정

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

전기 - 무료, 선형 핵산 및 단백질 분리

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

조직 해리 차동 여과 이식 미토콘드리아의 신속한 분리 및 정제

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

형광 DNA를위한 간단한 방법<em&gt; 현장에서</em&gt; 숙청 염색체에 하이브리드

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

디지털 핵산 정량 분석​​ 실험의 단순 대량 판독

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

세균 커뮤니티 특성화에 대한 효율적인 핵산 추출 및 16S rRNA의 유전자 시퀀싱

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

핵산의 NanoDrop Microvolume의 Quantitation

마이크로 볼륨 바이오 사양 나노 분광 광도계를 사용하여 핵산 및 단백질 농도 결정

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

전기 - 무료, 선형 핵산 및 단백질 분리

조직 해리 차동 여과 이식 미토콘드리아의 신속한 분리 및 정제

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

형광 DNA를위한 간단한 방법

디지털 핵산 정량 분석 실험의 단순 대량 판독

세균 커뮤니티 특성화에 대한 효율적인 핵산 추출 및 16S rRNA의 유전자 시퀀싱

세포 유래 세포 외 매트릭스의 빠른, 분리, 기능 연구에 대한 확장 방법, 및 분석

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

요약

더 많은 비디오 탐색

이 비디오의 챕터

핵산의 NanoDrop Microvolume의 Quantitation

마이크로 볼륨 바이오 사양 나노 분광 광도계를 사용하여 핵산 및 단백질 농도 결정

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

전기 - 무료, 선형 핵산 및 단백질 분리

조직 해리 차동 여과 이식 미토콘드리아의 신속한 분리 및 정제

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

형광 DNA를위한 간단한 방법

디지털 핵산 정량 분석​​ 실험의 단순 대량 판독

세균 커뮤니티 특성화에 대한 효율적인 핵산 추출 및 16S rRNA의 유전자 시퀀싱

세포 유래 세포 외 매트릭스의 빠른, 분리, 기능 연구에 대한 확장 방법, 및 분석

디지털 핵산 정량 분석 실험의 단순 대량 판독