Funding was provided by the Deutsche Forschungsgemeinschaft (SPP1230, grant of the Tumor Center Heidelberg/Mannheim), by the Bundesministerium f&#252;r Bildung und Forschung (iGene), by the VIth + VIIth Framework Programs of the European Commission (CONSERT, CLINIGENE and PERSIST). We thank Ina Kutschera for demonstrating the protocol technique in the video.

링커 카세트 1. 준비 (LC) <ol><li> LC1 올리고 뉴클레오티드 (표 1), (적절한 제한 효소 오버행 표 1) LC2 올리고, 110 ㎕의 트리스 - 염산 (100 ㎜, 산도 7.5), 10 ㎕의 250 mM의 MgCl2를 40 μL의 40 μl를 섞는다. </li><li> 5 분 95 ° C에서 품어 반응이 실온으로 천천히 냉각 할 수 있습니다. 300 μL의 H 2 O를 추가하고 원심 분리 필터에 dsLinker-DNA를 집중한다. 0.2 PCR 튜브에 준비 링커 카세트의 용출액과 나누어지는 10 μL에 80 μL의 H 2 O를 추가합니다. </li></ol> 벡터 게놈 접합 2. 프리 앰프 <ol><li> 분석 할 수있는 모든 샘플은 50 ㎕의 PCR 반응을 준비합니다. <ol><li> DNA 샘플의 농도를 결정합니다. 피펫은 X μL (1-1,000이 nrLAM에 겨 LAM/100-1, 000 겨) 0.2 ML PCR 튜브에 DNA의. DNA의 양이 각각의 샘플과 RAN에서 동일해야합니다0.5-25 μL의 GE. </li><li> 표 2에 설명 된대로 PCR 마스터 믹스를 준비 믹스 (50 - X). 0.2 ML PCR 튜브에 각각의 DNA 샘플과 마스터 믹스 μL. </li></ol></li><li> PCR을 완료 한 후. 표 2에 예시 된 PCR 조건을 사용하여 프리 앰프를 벡터 게놈 접합 각 PCR 튜브를 다시 실행 PCR 프로그램의 Taq 중합 효소 μL 0.5을 추가합니다. PCR 제품은 -20 ° C에서 최대 4 일 ~ 장기 4 ° C에 저장할 수 있습니다 </li></ol> PCR 제품의 3. 자기 분리 <ol><li> 자석 구슬의 준비 <ol><li> 피펫 (20) 1.5 ML 튜브에 스트렙 타비 딘 코팅 자석 구슬의 μL (200 μg) 및 상온에서 자성 입자 분리기 (MPS)에 1 분간 노출. 상층 액을 버린다. </li><li> MPS에서 튜브를 제거하고 40 ㎕의 PSB / 0.1 % BSA (산도 7.5)에 자석 구슬을 재현 탁. 1 분 동안 MPS에 노출하고 상층 액을 버린다. 일단이 단계를 반복합니다. </li><li> 워시 무선 비즈3 M의 LiCl 용액 20 μL 번째 (3 M LiCl을, 10 mM 트리스 - 염산, 1 ㎜ EDTA)은 1 분 동안 MPS에 노출하고 상층 액을 버린다. 6 M의 LiCl 용액 (6 M LiCl을, 10 mM 트리스 - 염산, 1 ㎜ EDTA)의 50 μL에 재현 탁 구슬. </li></ol></li><li> 준비 자석 구슬의 50 μL와 2.2 단계에서 전체 PCR 반응을 섞는다. 적어도 실온에서 2 시간 동안 수평 진탕 기 (300 RPM)에 품어. 이 단계는 코팅 된 비즈 (구슬 DNA 복합체를) 스트렙 타비 딘에 바이오틴 PCR 제품의 결합을 할 수 있습니다. DNA 구슬 복잡한 하룻밤 진탕 배양 또는 최대 4 일 동안 4 ° C에 저장할 수 있습니다. </li><li> 실온에서 1 분 동안 MPS에 DNA 비드 복잡한 노출, 상층 액을 제거하고 100 μL의 H 2 O의 DNA 비드 복잡한을 재현 탁 즉시 LAM 또는 nrLAM을위한 5 단계에 대해 4 단계를 진행합니다. </li></ol> 4. LAM-절차 <ol><li> 이중 가닥 DNA (dsDNA) 합성 (LAM 전용) <ol><li> 1 분 및 표시에 대한 MPS 단계 3.3에서 DNA 비드 복잡한 노출카드 뜨는. 8.25 H 2 O의 μL, 1 μL 배 hexanucleotide 버퍼 0.25 μL의의 dNTPs (10 ㎜) 0.5 μL (2 U) 클레 나우 중합 효소를 추가합니다. 1 시간 동안 37 ℃에서 배양한다. </li><li> H 2 O의 90 μl를 추가하고 1 분 동안 MPS에 노출. 100 μL의 H 2 O에 뜨는에 resuspend DNA 비드 복잡한 폐기 </li></ol></li><li> 제한 다이제스트 <ol><li> 1 분 동안 MPS에 DNA 비드 복잡한을 노출하고 상층 액을 버린다. 8.5 μL의 H 2 O, 1 ㎕의 10 배 제한 효소 버퍼와 제한 효소의 0.5 μl를 추가하고 1 시간 동안 반응을 품어. 4.1.2 단계를 반복합니다. 참고 : 제한 효소의 제조업체에서 권장하는 온도에서 반응을 품어. 제한 사이트 내에 존재하는 또는 관심의 DNA에 프리 앰프에 사용되는 프라이머 결합 부위의 다운 스트림이 없는지 확인합니다. 적당한 제한 효소 / 제한 효소 조합의 선택을 위해 9를 참조하십시오. </li></ol></li><li> 결찰 DS의 링커 (LK) <ol><li> 1 분 동안 MPS에 DNA 비드 복잡한을 노출하고 상층 액을 버린다. 5 μL의 H 2 O, 1 ㎕의 10 배 FastLink 버퍼, 1 μL의 ATP (10 ㎜), 단계 1.2 ~ 2 μL 링커 카세트, 1 ㎕를 고속 링크 DNA 리가 제 (2 U / μL)를 추가합니다. 5 분 동안 실온에서 배양한다. 4.1.2 단계를 반복합니다. </li></ol></li><li> 신시 dsDNA의 변성 <ol><li> 1 분 동안 MPS에 DNA 비드 복잡한을 노출하고 상층 액을 버린다. 5 ㎕를 0.1 N 수산화 나트륨에 재현 탁 DNA 비드 복잡한. 가로 통에 실온에서 5 분 알을 품다. </li><li> 1 분 동안 MPS에 DNA 비드 복잡한을 노출하고 새로운 1.5 ML 튜브에 뜨는을 포함 증폭 된 벡터 게놈 접합을 수집합니다. 즉시 -20 ℃에서 6 단계 또는 저장 상층 액을 진행 </li></ol></li></ol> 5. nrLAM - 절차 <ol><li> 단일 가닥 링커의 결찰 (SSLC) (nrLAM 만) <ol><li> MPS에 단계 3.3에서 DNA 비드 복잡한 노출1 분 및 폐기 뜨는하십시오. 6.5 μL의 H 2 O를 추가, 1 ㎕의 10 배 반응 버퍼, CircLigase 0.5 ㎕의 MnCl 2 (50 ㎜), 0.5 ATP (1 ㎜) μL, 1 μL의 ssLinker 올리고 0.5 μL의 CircLigase (100 U / μL). 1 시간 동안 60 ° C에서 알을 품다. </li><li> H 2 O의 90 μl를 추가하고 1 분 동안 MPS에 노출. 뜨는을 취소하고 100 μL의 H 2 O의 DNA 비드 복잡한 세척 다시 10 μL의 H 2 O에서 1 분, 폐기 뜨는에 resuspend DNA 비드 복잡한에 대한 MPS에 노출 </li></ol></li></ol> 6. 지수 증폭 I <ol><li> 분석 할 수있는 모든 샘플은 50 ㎕의 PCR 반응을 준비합니다. <ol><li> 피펫 2 μL 템플릿 (단계 4.4.2 (LAM부터) 또는 5.1.2 (nrLAM)) DNA 0.2 ml의 PCR 튜브에. </li><li> 표 3에 설명 된대로 PCR 마스터 믹스를 준비합니다. 단계 6.1.1에서 각각의 샘플에 마스터 믹스 48 μl를 추가하고 PCR 컨디셔닝 시스템에 의해 벡터 게놈 접합을 증폭표 3에 예시 된 NS는. PCR 제품은 -20 ° C에서 최대 4 일 ~ 장기 4 ° C에 저장할 수 있습니다 </li></ol></li></ol> PCR 제품의 7. 자기 분리 <ol><li> 3.1.3 - 3.1.1 단계에 예시 된 바와 같이 자석 구슬을 준비합니다. (LAM에 대한) 20 μL 6 M의 LiCl 용액 (6 M LiCl을, 10 mM 트리스 - 염산, 1 ㎜ EDTA)의 (nrLAM에 대한) 50 μL에 재현 탁 구슬. 20 μL (LAM) 또는 준비 자석 구슬 단계 6.1.2에서 PCR 반응 (단계 7.1) 50 μL (nrLAM)를 혼합하고 실온에서 2 시간 동안 수평 진탕 기 (300 RPM)에 품어. DNA 구슬 복잡한 하룻밤 진탕 배양 또는 최대 4 일 동안 4 ° C에 저장할 수 있습니다. </li><li> 1 분 MPS에 DNA 비드 복잡한 노출, 상층 액을 제거하고 100 μL의 H 2 O의 DNA 비드 복잡한을 재현 탁 1 분 동안 MPS에 DNA 비드 복잡한을 노출하고 상층 액을 버린다. </li><li> 20 μL (LAM) 또는 5 μL (nrLAM) 0.1 N 수산화 나트륨에 재현 탁 DNA 비드 복잡한. 인큐가로 통에 실온에서 10 분 동안 BATE는 1 분 MPS에 노출 새로운 1.5 ML 튜브에서 증폭 된 DNA가 포함 상층 액을 수집합니다. 즉시 -20 ℃에서 8.1 단계 또는 저장 상층 액을 진행 </li></ol> 8. 지수 증폭 II <ol><li> 분석 할 수있는 모든 샘플은 50 ㎕의 PCR 반응을 준비합니다. <ol><li> 0.2 ML PCR 튜브에 (단계 7.3) 피펫 2 μL 템플릿 DNA. </li><li> 표 4에 설명 된대로 PCR 마스터 믹스를 준비합니다. 단계 8.1.1에서 각각의 샘플에 마스터 믹스 48 μl를 추가하고 표 4에 예시 PCR 조건에 벡터 게놈 접합을 증폭. PCR 제품은 최대 4 동안 4 ° C에 저장할 수 있습니다 -20 ° C에서 일 또는 장기 </li></ol></li><li> 시각화 (NR) LAM-PCR 제품, 2 % 아가로 오스 겔상에서 단계 8.1.2의 PCR 생성물의 부하 10 μL. 밴드가 보이지 않는 경우, 고해상도 겔 LAM-PCR 생성물의 10 μl를 분석한다. 주의 : 잇hidium 브로마이드 변이원성이다. 매우 신중하게 작업하고 항상 적절한 장갑을 착용하십시오. </li></ol> 높은 처리량 시퀀싱 9. 준비 <ol><li> (NR) LAM-PCR 제품의 정제 <ol><li> 실내 온도 AMPure XP 자석 구슬의 44 μL와 단계 8.1.2에서 40 ㎕의 PCR-제품을 섞는다. 실온에서 5 분을 품어 추가로 2 분 동안 MPS에 노출. </li><li> 뜨는을 취소하고 MPS에 200 ㎕의 70 % EtOH로 두 번 씻어. </li><li> 30 μL의 H 2 O에 뜨는에 resuspend DNA 비드 복잡한 폐기 신선한 0.2 ML 튜브에 1 분 및 전송 뜨는을 품어. 정제 된 DNA의 농도를 결정합니다. </li></ol></li><li> Fusionprimer PCR은 특정 어댑터를 시퀀싱 추가 할 수 있습니다. <ol><li> 분석 할 수있는 모든 샘플은 50 ㎕의 PCR 반응을 준비합니다. </li><li> 0.2 ML PCR 튜브에 DNA의 피펫 X μL (40 NG). DNA의 양이 각각의 샘플과 0.5-25 μL의 범위에서 동일해야합니다. </li><li> 표 5에 설명 된대로 PCR 마스터 믹스를 준비 추가 (50 - X). 단계 9.2.2에서 각 샘플에 대한 마스터 믹스 ㎕의 표 5 PCR 제품에 예시 PCR 조건에 따라 (NR) LAM-PCR 제품 시퀀싱 어댑터를 소개합니다. -20 ° C에서 최대 4 일 ~ 장기 4 ° C에 저장할 수 있습니다 </li><li> Fusionprimer-PCR 제품 부하에게 2 % 아가 로스 겔상에서 단계 9.2.3의 PCR 생성물의 10 μL를 시각화. 9.1.3 - 9.1.1 단계에 설명 된대로 나머지 PCR 제품을 정화. 정확하게 Fusionprimer-PCR 제품의 농도 및 단편의 크기를 정량화하는 자동화 된 고해상도 전기 영동 장치에 단계 9.4에서 1 ㎕의 정제 된 PCR 생성물을 분석한다. </li></ol></li></ol>

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The authors have nothing to disclose.

LAM-PCR 기술은 공지 된 DNA 영역의 측면 불명 DNA 서열을 식별한다. 때문에 공지 된 DNA 서열에 특이 적 프라이머의 혼성화와 접합부의 증폭 량으로 인해 고감도, 그것은 단일 세포 수준까지조차 희귀 접합을 증폭 및 검출 할 수있다. 반대로, 클론 상황에서 LAM-PCR은 하나의 반응에 다른 접합의 수천을 증폭 할 수 있습니다. 그러나, 제한의 사용으로 인해이 integrome 만 하위 성분마다...

선형 증폭 PCR (LAM-PCR)을 매개하는 모든 근원의 알려진 DNA에 인접한 알 수없는 측면에서는 DNA를 식별하고 특성화 할 수 있습니다. 보다 구체적으로, LAM-PCR은 숙주 게놈 내에서 1,2 (IS) 바이러스 벡터의 삽입 부위를 집중하기 위해 개발되었다. 레트로 바이러스 나 트랜스포존과 같은 유전 적 요소는 (반) 임의의 방식 3-6 호스트 게놈에 자신의 게놈을 통합 할 수 있습니다. 많은 경우에 이러한 벡터가 통합 정확하게 위치를 알고 결정적이다. LAM-PCR은 결찰 매개 PCR 7 및 그 변종 또는 역 PCR 8와 같은 대체 기술에 우수한 것으로 입증되었습니다. 이 방법의 감도와 견고성은 벡터 게놈 접합 증폭 PCR 제품의 자기 선택의 최초의 프리 앰프에서 발생한다. 언급 된 다른 방법과 마찬가지로, LAM-PCR은 9-11의 검색 능력에 편견을 도입, 제한 효소의 사용에 의존한다. 따라서,IS 레퍼토리 (integrome)의 서브 세트 만이 하나의 반응에서 검출 될 수있다. 이 바이어스는 제한 효소 (9)의 최적의 조합을 사용하여 주어진 샘플의 병렬 분석을 통해 최소화된다. 최근 기술의 변형은 제한 효소의 사용을 우회 한 번의 반응 9,12있는 샘플의 공정한 게놈 넓은 해석을 허용하는 LAM-PCR (nrLAM-PCR)이 개발되었다 비 제한 칭했다. 과거에는, LAM-PCR은 원인 레트로 바이러스가 유전자 치료 임상 시험 13 ~ 15에서 몇 환자에서 백혈병에 상승을주고 식별하는 데 사용되었습니다. 그 이후로, LAM-PCR을 식별하기에 적합 한 다른 통합 벡터 (렌티 바이러스 벡터, 트랜스포존) 또한 수동적으로 아데노 - 관련 벡터 (AAV) 또는 인테그라 불량 렌티 바이러스 벡터 (IDLV)와 같은 벡터를 통합하는 통합 패턴을 식별 할 수에서 (16) -21. LAM-PCR의 응용 프로그램은 광범위한 확산되어 기존의LY,이 기술은 널리 유전자 치료를받은 환자에서 유전자 변형 세포의 클론 구성을 공부하거나 통합 문제 15,16,22-24을 탈피하여 새로운 벡터 시스템의 바이오 안전성을 평가하는 데 사용됩니다. 최근, LAM-PCR은 IDLV 트래핑 분석 (25)에 의해 특이 디자이너 클레아의 오프 대상 활동을 결정하는 사용. 또한, LAM-PCR 쉽게 유기체에서 시간이 지남에 형질 세포의 운명을 수행 할 수 있습니다. 이 프로토 암 유전자뿐만 아니라 종양 억제 유전자를 확인하고 또한 조혈 또는 암 줄기 세포 생물학 26-28을 연구 할 수 있습니다. 마지막으로 적어도, LAM-PCR은 인간 29 (및 게시되지 않은 데이터)에 T-세포 수용체의 다양성을 연구하기 위해 적응했다. 기술의 본질적인 힘은 단일 염기 R과 함께 알 수없는 측면에서는 DNA의 수백만의 특성을 허용 깊은 시퀀싱 기술과 방법을 연결하여 강화전체 게놈의 esolution. 다음과 같은 프로토콜에서, 우리는 단계별로 증폭 및 렌티 바이러스 벡터입니다 확인하는 예시 적으로 알 수없는 DNA를 측면에서는 식별을 설명합니다. 프로토콜에 사용 된 올리고 뉴클레오티드. 어떤 소스의 추출 된 DNA 또는 cDNA를이 LAM-PCR 및 nrLAM-PCR을위한 DNA 템플릿으로 이용 될 수는 표 1에 나타내었다.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:120px;">Catalog Number</td>
			<td style="width:527px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Taq DNA Polymerase</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3001.5000</td>
			<td>Alternative Taq Polymerases may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR Buffer</td>
			<td style="width:203px;">Qiagen</td>
			<td style="width:120px;">201203</td>
			<td>Use of this buffer is recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">dNTP-Mixture</td>
			<td style="width:203px;">Genaxxon Bioscience GmbH</td>
			<td style="width:120px;">M3015.4020</td>
			<td>or any other dNTPs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Oligonucleotides (Primers)</td>
			<td style="width:203px;">MWG Biotech</td>
			<td style="width:120px;"></td>
			<td>HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dynabeads M-280 Streptavidin&#160;</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">11206D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PBS</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:120px;">14190-086</td>
			<td>0.1 % wt/vol BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">6M LiCl</td>
			<td style="width:203px;">Roth</td>
			<td style="width:120px;">3739.1</td>
			<td>10 mM Tris-HCl&#160; (pH 7.5)/1 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tris-HCl, pH 7.5</td>
			<td style="width:203px;">USB Corporation&#160;</td>
			<td style="width:120px;">22637</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">EDTA</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A1103,0250</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Klenow Polymerase</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">10104523001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Hexanucleotide mixture</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11277081001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Restriction endonuclease</td>
			<td style="width:203px;">NEB</td>
			<td style="width:120px;"></td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Fast-Link DNA ligation kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">LK11025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CircLigase ssDNA Ligase Kit</td>
			<td style="width:203px;">Epicentre Biotechnologies</td>
			<td style="width:120px;">CL4111K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">NaOH</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">72068</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Agarose LE</td>
			<td style="width:203px;">Roche Diagnostics</td>
			<td style="width:120px;">11685660001</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TBE buffer</td>
			<td style="width:203px;">Amresco</td>
			<td style="width:120px;">0658</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ethidium bromide</td>
			<td style="width:203px;">Applichem</td>
			<td style="width:120px;">A2273,0005</td>
			<td>Ethidium bromide is mutagenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">100 bp DNA Ladder</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:120px;">15628-050</td>
			<td>or any other DNA ladder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">20 mM NaCl</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:120px;">71393-1L</td>
			<td>or any other supplier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158501</td>
			<td>for use with 1.5 ml Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Magna-Sep Magnetic Particle Separator&#160;</td>
			<td style="width:203px;">Life Technologies</td>
			<td style="width:120px;">K158696</td>
			<td>for use with 96 well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-0.5, Ultracel-30 membrane</td>
			<td style="width:203px;">Millipore</td>
			<td>UFC503096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PerfectBlue Gelsystem Midi S</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;">40-1515</td>
			<td>or other electrophoresis system&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TProfessional 96</td>
			<td style="width:203px;">Biometra</td>
			<td style="width:120px;">050-551</td>
			<td>or other Thermocycler for 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Orbital shaker KS 260 basic</td>
			<td style="width:203px;">IKA</td>
			<td style="width:120px;">2980200</td>
			<td>or other horizontal shaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">PCR softtubes 0.2 ml</td>
			<td style="width:203px;">Biozym Scientific GmbH</td>
			<td style="width:120px;">711082</td>
			<td>or other 0.2 ml PCR tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">1.5 ml tubes</td>
			<td style="width:203px;">Eppendorf</td>
			<td style="width:120px;">12682</td>
			<td>or other 1.5 ml tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Gel documentation system</td>
			<td style="width:203px;">PeqLab</td>
			<td style="width:120px;"></td>
			<td>or any other gel documentation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Nanodrop ND-1000 spectrophotometer</td>
			<td style="width:203px;">Thermo Scientific</td>
			<td style="width:120px;">ND-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Spreadex EL1200 precast gel</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">3497</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:360px;">Submerged gel electrophoresis apparatus SEA 2000&#160;</td>
			<td style="width:203px;">Elchrom Scientific</td>
			<td style="width:120px;">2001E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">2100 Electrophoresis Bioanalyzer</td>
			<td style="width:203px;">Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
	</tbody>
</table>

linear amplification mediated pcr &#8211; localization of genetic elements and characterization of unknown flanking dna

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

LAM-PCR 각 접합에 대한 정의 조각의 크기와 벡터 게놈 접합의 증폭 결과. 각각의 PCR 단편의 크기는 게놈 DNA의 공지 된 위치와 가장 가까운 제한 효소 인식 부위 사이의 거리에 의존한다. 이 겔 전기 영동에 의해 분석 샘플 예에서 증폭 접합 다양성을 시각화한다. 단지 단일 (모노클로 날), 몇개 (올리고 클론), 또는 다수의 (폴리 클로 날) 밴드는 겔에 존재하는 경우. LAM-PCR의 결과는 가장 높은 ?...

Watch this Scientific Journal Video about Linear Amplification Mediated PCR â€“ Localization of Genetic Elements and Characterization of Unknown Flanking DNA at JoVE.com

Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

선형 증폭 중재 (LAM)-PCR은 게놈에 바이러스 벡터를 통합의 정확한 위치를 식별하기 위해 개발 된 방법입니다. 이 기술은 유전자 치료 환자 등 새로운 벡터 기술, T-세포의 다양성, 암 줄기 세포 모델의 바이오 안전성에 클론 역학을 공부하는 우수한 방법으로 진화하고있다

선형 증폭 매개 PCR - 알 수없는 측면에서는 DNA의 유전 적 요소와 특성의 현지화

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

linear-amplification-mediated-pcr-localization-genetic-elements

Research

JoVE Journal

Biology

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

29.9K 조회수.  National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ).  선형 증폭 중재 (LAM)-PCR은 게놈에 바이러스 벡터를 통합의 정확한 위치를 식별하기 위해 개발 된 방법입니다. 이 기술은 유전자 치료 환자 등 새로운 벡터 기술, T-세포의 다양성, 암 줄기 세포 모델의 바이오 안전성에 클론 역학을 공부하는 우수한 방법으로 진화하고있다 

비디오: Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

연구

생물학

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

교양 차 마우스 배아 섬유아 세포의 신호 분자의 아데노 바이러스 - 매개 유전자 제거

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

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 ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

의 유전자 표현 프로필 결정 C. 엘레간스 Microarray 및 실시간 PCR을 사용하여

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

단백질-DNA 상호 작용의 연구에 대한 단일 분자 조작 및 이미지를 결합

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

DNA 일렉트로, 근섬유의 분리 및 이미징

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

쥐 간에서 DNA 가닥 나누기 및 산화 적 DNA 손상을 측정하기위한 생체 알카라인 혜성 분석 및 효소 수정 알카라인 혜성 분석에서

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

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 ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.