Name: methyl-binding dna capture sequencing for patient tissues
Uploaded: 2016-10-31T12:30:00.000+00:00
Duration: 8 min 40 s
Description: Watch this Scientific Journal Video about Methyl-binding DNA capture Sequencing for Patient Tissues at JoVE.com

이 작품은 CPRIT 연구 교육 상 RP140105에서 지원뿐만 아니라 부분적으로 건강의 미국 국립 보건원 (NIH) R01 GM114142을 부여하여 윌리엄 &amp; 엘라 오웬스 의학 연구 재단이 지원됩니다.

모든 조직은 임상 시험 심사위원회위원회의 승인을 다음과 얻어진 모든 참가자가 분자 분석과 후속 연구에 모두 동의 할 때. 프로토콜은 샌 안토니오 텍사스 건강 과학 센터의 대학에서 인간 연구위원회에 의해 승인됩니다. 1. 메틸 결합 DNA 캡처 (MBDCap) <ol><li> 샘플 수집 및 DNA 분리 <ol><li> 환자 파라핀 조직 샘플에서 대량 종양 또는 정상 조직 샘플을 수집합니다. </li><li> 제조 업체의 프로토콜에 따라 파라핀 조직 샘플에서 게놈 DNA를 분리 상업 DNA 미니 키트를 사용합니다. </li><li> 제조 업체의 프로토콜에 따라 여기에 설명 된 절차에 메틸화 DNA 농축 키트를 사용합니다. </li></ol></li><li> 초기 비드 세척 참고 : 구슬은 게놈에 DNA 메틸화를 검출 MBD - 비오틴 단백질과 커플하는 데 사용됩니다. 구슬은 일반적으로 일시 중단주식 솔루션입니다. 이 액체를 씻어하려면 다음 단계를 사용합니다. <ol><li> 균일 한 현탁액을 얻기 위해 아래로 부드럽게 피펫 팅에 의해 스트렙 타비 딘 비즈 (재료 표 참조)의 주식을 재현 탁합니다. 텍싱하여 구슬을 혼합하지 마십시오. </li><li> 입력 DNA 중 하나 μg의 경우, 1 배 바인딩 / 워시 버퍼의 90 μL와 튜브 구슬의 add10 μL. 1 분 동안 회전합니다. 텍싱하여 혼합하지 마십시오. </li><li> 1 분 동안 자기 랙에 튜브 (들)을 놓습니다. </li><li> 피펫으로 액체를 제거하고 액체를 폐기합니다. </li><li> 구슬에 1 배 바인딩 / 워시 버퍼 250 μL를 추가하고 1 분 동안 회전합니다. </li><li> 두 번 1.2.5 - 반복 1.2.3 단계를 반복합니다. </li></ol></li><li> 비드에 MBD-비오틴 단백질 커플 링 <ol><li> 입력 DNA의 1.2 μg의, 각 튜브에 MBD - 비오틴 단백질의 7 μL (3.5 μg의)를 추가합니다. </li><li> 100 μL의 최종 볼륨을 만들기 위해 단백질 1 배 바인딩 / 워시 버퍼의 93 μL를 추가합니다. </li><li> 구슬 단백질을 혼합실온에서 1 시간 동안 회전 혼합기에서 혼합. </li></ol></li><li> 단편화 된 DNA (입력) <ol><li> TE 버퍼 96 μL, 24 μL 5 배의 귀속 / 세척 버퍼에 전체 게놈 DNA의 1.2 μg의를 추가하여 초음파 처리 DNA 120 μL 솔루션을 확인합니다. 25 시간 - 30의 전원 ON 및 초음파 동안 30의 전원 OFF, 20을 사용합니다. </li><li> 제조사의 프로토콜에 따라 혈압 (350) - (150)의 범위로 단편의 크기를 확인하는 상업용 고감도 DNA 키트와 bioanalyzer 시스템을 이용하여 초음파 DNA 용액 1 μL를 실행. </li></ol></li><li> MBD-구슬을 씻으십시오 <ol><li> 1 분 동안 자기 선반에 MBD-구슬이 들어있는 튜브를 놓습니다. </li><li> 제거하고 구슬을 건드리지 않고 피펫으로 액체를 폐기합니다. </li><li> 1 배 바인딩 / 워시 버퍼 250 μL와 비드를 재현 탁. </li><li> 5 분 동안 실온에서 회전 믹서 비드 섞는다. </li><li> 두 번 1.5.4 - 반복 1.5.1 단계를 반복합니다. </l난&gt; </ol></li><li> 단편화 된 DNA 캡처 반응 (1 μg의 입력 DNA) <ol><li> MBD 비드를 함유하는 튜브에 DNA / 완충액 혼합물을 전송. </li><li> RT에서 1 시간 동안 회전 혼합기에서 DNA와 MBD 비드 섞는다. 또한, 4 오 ℃에서 O / N을 혼합 </li></ol></li><li> 구슬에서 비 캡처 DNA 분리 <ol><li> 1 분은 상기 튜브의 내벽에 비드 모두 집중하기 위해 DNA와 MBD 비드를 혼합 한 후, 자기 선반에 튜브를 놓는다. </li><li> 피펫으로 상층 액을 제거하고 DNA nonmethylated 같은 깨끗한 DNase의 프리 microcentrifuge 관에 저장한다. 얼음이 샘플을 저장합니다. </li><li> 구슬을 씻어 구슬에 1 배 바인딩 / 워시 버퍼 200 μL를 추가합니다. </li><li> 3 분 동안 회전 믹서 비드 섞는다. </li><li> 1 분 동안 자기 랙에 튜브를 놓습니다. </li><li> 피펫으로 액체를 제거하고 microcentrifuge 관에 저장합니다. </li><li> 캡총이 더 세척 분획을 위해 1.7.6 - 입력 DNA의 ≤1 μg의의 반응을 진짜야, 반복 1.7.3 단계를 반복합니다. </li></ol></li><li> 단일 분수 용출 <ol><li> 높은 소금 버퍼 저염 버퍼 믹스 (1 : 1) 용출 버퍼 (1,000 밀리미터의 NaCl)을 얻을 수 있습니다. </li><li> 용출 버퍼 (1000 mM의 염화나트륨)의 200 μL의 비드를 재현 탁. </li><li> 3 분 동안 회전 믹서에 구슬을 품어. </li><li> 1 분 동안 자기 랙에 튜브를 놓습니다. </li><li> 자기 선반에 장소에 튜브, 피펫 팁으로 구슬을 건드리지 않고 피펫으로 액체를 제거하고 깨끗한 DNase의 프리 1.5 ML의 microcentrifuge 관에 저장합니다. 얼음이 샘플을 저장합니다. </li><li> 반복 1.8.1 단계 - 같은 튜브의 두 번째 샘플을 수집 한 번 1.8.4을 (총 부피는 400 μL 될 것입니다). 얼음에 풀링 된 샘플을 저장합니다. </li></ol></li><li> 에탄올 침전 (DNA 정리) <ol><li> 각 noncaptured, 세척 및 용출 분수 FR에이전 단계 OM 1 μL의 글리코겐을 추가 (20 μg의 / 키트에 포함 μL), 3M 아세트산 나트륨 1/10 샘플 볼륨의 pH 5.2 (예를 들어, 400, 40 μL 샘플 μL) 및 100 %의 2 샘플 볼륨 에탄올 (예, 400 당 800 μL 샘플 μL). 총 부피는 1241 μL이다. </li><li> 잘 믹스 적어도 2 시간 동안 C O를 -80에서 품어. </li><li> 4 오 ℃에서 11,363 XG에 15 분 동안 튜브를 원심 분리기 </li><li> 조심스럽게 펠렛을 방해하지 않고 상층 액을 버린다. </li><li> 차가운 70 % 에탄올 500 μL를 추가합니다. </li><li> 4 오 ℃에서 11,363 XG에서 5 분 튜브를 원심 분리기 </li><li> 조심스럽게 펠렛을 방해하지 않고 상층 액을 버린다. </li><li> 1.9.7 한 번 남아있는 잔류 뜨는을 제거 - 반복 1.9.6 단계를 반복합니다. </li><li> ~ 5 분 펠렛을 공기 - 건조. 완전히 펠렛을 건조하지 마십시오. </li><li> DNase의없는 물 또는 기타 37.5 μL의 DNA 펠렛을 재현 탁버퍼의 적절한 볼륨. </li><li> 얼음의 DNA를 배치 또는 -20 ℃로 또는 아래에서 더 사용할 때까지 DNA를 저장합니다. </li><li> DNA의 총량이 20 ng의 위에 있는지 확인합니다 (예, 형광 정량 시스템을 사용, 재료 표 참조). </li></ol></li></ol> 2. 시퀀싱 <ol><li> 시퀀싱 MBDCap 절차에서 회수 된 DNA 조각을 사용합니다. 각 샘플 시퀀싱하기 위해서는 (20)의 총량 - 40 ng의 DNA를 라이브러리 제조를 위해 요구된다. </li><li> DNA-SEQ 라이브러리 준비는 완전 자동화 된 라이브러리 구축 시스템을 사용하십시오 (재료 표 참조) 및 제조 업체에서 제공하는 표준 프로토콜을 따르십시오. 라이브러리 준비를위한 400 bp의 - 크기 (200)의 선택 DNA 조각. 자동화 라이브러리 제조 시스템은 라이브러리 제조 과정을 표준화 의한 수동 제제에 샘플에 걸쳐 변화를 최소화 허용한다. </li><li> 사용 어댑터 프라임RS 및 100 배 농도로 희석. 이 중, 각 샘플 10 μL를 사용합니다. </li><li> 단계 2.2에 따라, 반응을 수행하고, 형광 정량 시스템 (재료 표 참조)를 사용하여 DNA-SEQ 라이브러리를 정량화. <ol><li> PCR 증폭 과정을 설정합니다. PCR 마스터 믹스의 선택은 게놈의 GC 풍부 영역의 PCR 효율을 향상시키는 것이다. PCR의 관에서 15 μL DNA-서열 라이브러리를 추가, 25 μL PCR 마스터 믹스 (재료 표 참조) 2 μL PCR 프라이머 믹스 (재료 표 참조), 8 μL H 2 O </li></ol></li><li> 출발 물질을 다른위한 순환 시간과 온도 변화의 PCR 마스터 믹스의 제조 업체에서 PCR 권고를 따라 98 O C 45 초 동안, 15 초 98 O를 C, 30 초 65 O를 C, 30, 72 O를 C의 X 사이클 의, 1 분 후 72 O를 C. 4 오 ℃에서 잡아 <ol><li> 반환 한(- 10 회 8) 라이브러리 DNA의 10 nm의 - 2로 끝날 정도로 사이클을 ORM. </li></ol></li><li> 1 PCR 반응 정리 : 제조 업체의 프로토콜에 따라 PCR 정화 구슬의 1 비율 (재료 표 참조). </li><li> 30 μL 용출 완충액 - 20 용리. </li><li> 흐름 셀의 한 차선으로 4 샘플 함께 각각의 샘플과 풀을 바코드. 시퀀싱에 대한 표준 50 bp의 단일 읽기 프로토콜 (재료 표 참조)를 사용합니다. </li></ol> 3. 생물 정보학 분석 주의 : 서열로부터 수득 또한 프로세스 원료 fastq 파일은 품질 제어를 수행하며 게놈 짧은 DNA 서열 (판독) 매핑. <ol><li> 사용 나비 넥타이 짧은 판독 노광 각 샘플 fastq 파일의 게놈 맵핑 도구가 기준 게놈 판독 매핑한다. 다수의 판독 매핑 툴이 단계를 수행 할 수있는 개인 선호도에 따라 사용될 수있다. <ol><li> 도 ë 2 불일치까지 허용참조 게놈에 매핑하는 동안 전자는 읽습니다. 각 읽기 (20) 최고의 정렬까지보고합니다. 예를 들어, 나비 넥타이 -v 2 --best -k 20 --chunkmbs 200 &lt;ebwt&gt; &lt;fastqFile&gt; &lt;alignFile&gt;를 사용합니다. </li></ol></li><li> 다중 매핑이 농후 영역의 검출을 개선하기 위해 판독 복구 LONUT 9를 사용한다. 각각의 판독의 경우, 최대 하나의 배향으로는 상기 호출 피크 중 충분히 근접 할 때 복원 될 것이다. 예를 들어, 사용 : 펄 lonut.pl -g hg19 -w (250) -p (99) &lt;alignFile&gt; &lt;outputPath&gt;. LONUT 다음 단계를 수행합니다. <ol><li> 로 분할 정렬 고유 읽고 매핑과 다중 읽기 매핑. 유일하게 매핑에 전화 피크는 피크 발신자 벨트 (11)를 사용하여 읽습니다. 예제 명령 (250) 및 -p 99 -w 옵션은 BELT위한 것입니다. </li><li> 고유 매핑 수확기는 LONUT에서 얻은 읽기 회복과 함께 읽고, 결합은 침대 형식으로 추가 분석에 사용됩니다 읽습니다. COMBIN 수를 계산에드는 미래의 정상화를 위해 읽습니다. </li></ol></li><li> 빈 읽습니다. 추출 펄 스크립트를 사용하여 관심의 영역에서 읽 펄 methyPipeline.pl &lt;sampleInfoFile&gt; &lt;refGeneFile&gt; - 최대 &lt;상류 연장 길이&gt; -dn &lt;하류 연장 길이&gt;. sampleInfoFile에 나열된 각 침대 파일의 경우, 펄 스크립트는 아래의 작업을 수행합니다. 참고 : 펄 스크립트 추가 코딩 파일을 참조하십시오. <ol><li> 빈 24 염색체를 들어, 고정 빈 크기로 예를 들어, 100 bp의를 읽습니다. </li><li> refGeneFile에 지정된 각 유전자의 경우, 옵션 - 최대 및 -dn에 의해 지정된 연장 길이까지 전사 시작 사이트 주위의 빈 (TSS)을 추출한다. 예를 들어, 최대 및 다운 스트림 TSS 4 KB입니다 지역에서 데이터를 추출합니다. 빈 크기는 100 염기쌍, 이것은 데이터가 81 빈들을 추출하고, 상기 중앙의 빈 TSS에 대응한다. </li><li> 왼쪽에서 오른쪽으로 상류 쓰레기통은 항상 있도록 안티센스 가닥의 유전자를 들어, 괞 찮아 추출 된 영역을 반전상기 펄 스크립트 왼쪽에 세드릭. </li><li> 2킬로바이트을 상류 왼쪽, 상류 4킬로바이트; 또한 세 개의 하위 영역으로 TSS ± ​​4킬로바이트 영역을 분할 중동 최대 및 다운 스트림 2킬로바이트; 오른쪽, 4킬로바이트에 2킬로바이트 하류. </li><li> 3.3.3 계산 판독 매핑를 합한 총 횟수로 각 빈 판독의 각 샘플에 대해, 상기 카운트를 나눈다. 샘플 특정 제거됩니다 정규화는 차이를 읽고 다른 샘플에서 읽고 비교할 수 있습니다. </li></ol></li><li> 왼쪽 영역으로부터 얻어진 판독 정규화에서 통계 테스트를 수행 (스크립트 파일에 나타낸 바와 같이) 측면 영역으로 기술 영역에서 정상적인 경우 그룹간에 차이 메틸화를 검출하는 상기 배시 스크립트를 실행. 주 : bash는 스크립트의 추가 코딩 파일을 참조하십시오. 차별적 메틸화 된 지역을 기반으로, 일곱 조합이있다 : 지역 전체 다음 TSS ± ​​4킬로바이트 영역에 걸쳐 차동 메틸화 MiddleLeft : 모두 중앙과 왼쪽 지역에서 차동 메틸화 MiddleRight : 모두 중간과 오른쪽 지역에서 차동 메틸화 측면 : 왼쪽과 오른쪽 지역에서 차동 메틸화 왼쪽 : 왼쪽 지역에서 차동 메틸화 만 오른쪽 : 오른쪽 지역에서 차동 메틸화 만 중간 : 중간 영역에 차등 메틸화 만 </li><li> 토네이도 플롯에서 차동 메틸화를 시각화하기 위해 동일한 bash는 스크립트를 사용합니다. 별도의 패널에 하이퍼 및 하이포 메틸화 유전자를 플롯 각각 3.4에 설명 된 영역의 조합의 순서로 정렬. </li></ol>

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이 프로토콜은 샌 안토니오 텍사스 건강 과학 센터의 대학에서 박사 팀 황 박사 빅터 진의 실험실에서 개발되고있다.

MBDCap-SEQ 기술은 환자 (15)의 다수 집단을 조사 할 때 비용 효율적인 대안으로 고려 친 화성 농축 방법 (3)이다. 여기에 제시된 파이프 라인은 데이터 분석 및 해석에 샘플 조달에서 포괄적 인 접근 방식을 설명합니다. 가장 중요한 단계 중 하나는 DNA 메틸화가 발생하는 곳이므로 게놈에서 GC 풍부 영역의 PCR 효율을 향상시키기 위해 PCR 증폭 과정을 설정한다. 또한, 시퀀싱 후, 각각의 샘플이 유일하게...

DNA 메틸화를 통해 유전자의 후생 유전 학적 조절은 본체 (1)의 서로 다른 조직 유형의 안정에 의해 분화 세포의 운명을 결정하기 위해 필요한 필수 메커니즘이다. 이 과정의 이상 조절이 암을 비롯한 다양한 질환을 일으키는 것으로 알려져있다. 이 프로세스는 주로 DNA (3)의 CpG 디 뉴클레오티드의 시토신 잔기에 메틸기의 첨가를 포함한다. 현재 많은 연구 2-8에서 설명한 바와 같이 각각 자신의 장점을 갖는 이러한 메카니즘을 조사하기 위해 사용되는 몇 가지 방법이있다. 여기에서 우리는 우리가 DNA의 메틸화 지역을 식별하는 친 화성 농축 기술을 사용 (MBDCap-SEQ) 메틸 바인딩 DNA 캡처 순서라는 이러한 기술 중 하나를 설명합니다. 이 기술은 메틸화 된 CpG 부위를 함유하는 게놈 DNA 단편에 대한 농축하는 MBD2 단백질 메틸 결합능에 만든다. 우리는 상업 메틸화 된 DNA 농축 키트를 활용이러한 메틸화 영역의 분리를위한. 우리의 실험은이 기술을 사용하여 환자 샘플 수백 스크리닝하고 여기 큰 환자 집단을 조사하는데 사용될 수있는 최적화 된 광범위한 프로토콜을 제공한다. 모든 차세대 시퀀싱 기술 명백한 바와 같이, MBDCap-SEQ 또한 정확하게 샘플 통해 메틸화의 수준을 정량화하기 위해 특정 바이오 인포 매틱스 방법을 필요로한다. 시퀀스 데이터 (9), (10)의 정규화 및 분석 프로세스를 최적화하기 위해 많은 최근의 연구가 있었다. LONUT - - 환자 샘플의 다수에 걸쳐 편향 비교를 가능하게하기 위해, 각 샘플의 선형 정규화 뒤에이 프로토콜에서는 고유 판독 복구 방식을 구현하는 방법 중 하나를 보여준다.

<table><tbody><tr><td>Methylminer DNA enrichment Kit</td><td>Invitrogen</td><td>ME10025</td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin</td><td>Invitrogen</td><td>112-05D</td><td></td></tr><tr><td>Bioruptor Plus Sonication Device</td><td>diagenode</td><td>B01020001</td><td></td></tr><tr><td>3 M sodium acetate, pH 5.2</td><td>Sigma</td><td>S7899</td><td>100 mL</td></tr><tr><td>SPRIworks Fragment Library System I</td><td>Beckman Coulter</td><td>A50100</td><td>Fully automated library construction system</td></tr><tr><td>Adapter Primers</td><td>Bioo Scientific</td><td>514104</td><td>PCR primer mix</td></tr><tr><td>Qubit</td><td>Invitrogen</td><td>Q32854</td><td>Fluorometric Quantitation System</td></tr><tr><td>PCR master mix</td><td>KAPA scientific</td><td>KK2621</td><td>PCR master mix</td></tr><tr><td>AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>PCR Purification beads</td></tr><tr><td>EB Buffer</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>HiSeq 2000 Sequencing System</td><td>Illumina</td><td></td><td></td></tr></tbody></table>

methyl-binding dna capture sequencing for patient tissues

메틸화 다른 조직 유형의 안정 발생과 분화에 필요한 유전자의 정확한 조절을 담당하는 DNA에 필수적인 후생 변형 중 하나이다. 이 과정의 이상 조절은 종종 암과 같은 다양한 질환의 특징이다. 여기서, 우리는 최근 시퀀싱 기술을 간략 메틸 - 바인딩 많은 환자 집단에 대한 다양한 정상 및 질병 조직에 메틸화를 정량화하는 데 사용되는 캡쳐 DNA 시퀀싱 (MBDCap-SEQ)를. 우리는 최적의 정량화를 달성하기 위해 생물 정보학 파이프 라인과 함께이 친 화성 농축 방법의 상세한 프로토콜을 설명합니다. 이 기술은 1000 methylome 프로젝트 (암 Methylome 시스템)의 일부로서 다양한 종류의 암 환자에 걸쳐 수백 시퀀스 사용되고있다.

우리는 그 중에서도 유방 12, 13, 자궁 내막, 전립선 (14), 및 간암 등 다양한 종류의 암 환자에서 다수의 DNA 메틸화 변화를 연구 MBDCap - 배열을 사용했다. 여기에서 우리는 최근에 12 게시 된 유방암 연구에서 몇 가지 정보를 보여줍니다. 이 예에서, 우리는 다른 차등 게놈 영역에 걸쳐 정상에 대해 종양 메틸화 된 CpG 섬을 식별 총 유전체 방식을 사용 하였다. 조사는 개 가운데 유전자 ...

Watch this Scientific Journal Video about Methyl-binding DNA capture Sequencing for Patient Tissues at JoVE.com

Methyl-binding DNA capture Sequencing for Patient Tissues

여기서 우리는 프로토콜이 메틸 바인딩 DNA 캡처 시퀀싱 (MBDCap-서열 또는 MBD-SEQ) 기술과 이후의 생물 정보학 분석 파이프 라인을 사용하여 대규모 임상 환자 스크리닝 연구에서 게놈 전체 DNA 메틸화를 조사하기 위해 제시한다.

환자 조직에 대한 메틸 결합 DNA 캡처 시퀀싱

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

methyl-binding-dna-capture-sequencing-for-patient-tissues

Research

JoVE Journal

Genetics

8.6K 조회수.  University of Texas Health Science Center at San Antonio.  여기서 우리는 프로토콜이 메틸 바인딩 DNA 캡처 시퀀싱 (MBDCap-서열 또는 MBD-SEQ) 기술과 이후의 생물 정보학 분석 파이프 라인을 사용하여 대규모 임상 환자 스크리닝 연구에서 게놈 전체 DNA 메틸화를 조사하기 위해 제시한다. 

비디오: Methyl-binding DNA capture Sequencing for Patient Tissues

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

만성 림프 성 백혈병 환자에서 Methyl-CpG- 결합 도메인 포착 기반 방법을 이용한 포괄적 인 DNA 메틸화 분석

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

연구

유전학

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

미토 콘 드리 아 DNA 메 틸 화의 정확한 탐지를 위한 방법론

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Methylated DNA의 Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

생물학

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

뇌의 작은, 해부학적인 몸의 구조 정의 분야의 DNA Methylation과 유전자 발현의 최적화 분석

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

신경과학

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

위장암에서 DNA 메틸화의 게놈 전체 분석

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

암 연구학

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

의 DNA Methylation : Bisulphite의 변경 및 분석

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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 ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

In Vitro (시험관 ) 표적 후성유전학적 침묵을 위한 엔지니어링된 전사 억제제 선택

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

생체공학

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

자료 쌍 해상도에서 DNA 메틸화의 평가에 대한 표현 바이 설 파이트 시퀀싱을 감소 향상

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

DNA 메틸화 분석 차세대 시퀀싱 대상

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...