The authors thank Colleen Van Kirk for mouse retina and cerebellum samples, and Peter Gregory for figure generation. The authors also thank Dr. Allison Gillaspy and the Laboratory for Molecular Biology and Cytometry Research core for access to the MiSeq. This work was supported by NIH grants DA029405, EY021716, and AG026607 and the Donald W. Reynolds Foundation.

<ol>
	<li>Hotchkiss, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantitative+separation+of+purines,+pyrimidines,+and+nucleosides+by+paper+chromatography.">The quantitative separation of purines, pyrimidines, and nucleosides by paper chromatography.</a> The Journal of Biological Chemistry. 175 (1), 315-332 (1948).</li><li>Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+patterns+and+epigenetic+memory.">DNA methylation patterns and epigenetic memory.</a> Genes &amp; Development. 16 (1), 6-21 (2002).</li><li>Smith, Z. D., Meissner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+roles+in+mammalian+development.">DNA methylation: roles in mammalian development.</a> Nature reviews. Genetics. 14 (3), 204-220 (2013).</li><li>Heard, E., Martienssen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenerational+epigenetic+inheritance:+myths+and+mechanisms.">Transgenerational epigenetic inheritance: myths and mechanisms.</a> Cell. 157 (1), 95-109 (2014).</li><li>Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+and+gene+silencing+in+cancer.">DNA methylation and gene silencing in cancer.</a> Nature Clinical Practice. Oncology. 2, S4-S11 (2005).</li><li>Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+epigenome:+its+origins,+contributions+to+tumorigenesis,+and+translational+implications.">The cancer epigenome: its origins, contributions to tumorigenesis, and translational implications.</a> Proceedings of the American Thoracic Society. 9 (2), 64-65 (2012).</li><li>Beck, S., Rakyan, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methylome:+approaches+for+global+DNA+methylation+profiling.">The methylome: approaches for global DNA methylation profiling.</a> Trends in Genetics : TIG. 24 (5), 231-237 (2008).</li><li>Laird, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+challenges+of+genomewide+DNA+methylation+analysis.">Principles and challenges of genomewide DNA methylation analysis.</a> Nature reviews. Genetics. 11 (3), 191-203 (2010).</li><li>Mikeska, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+quantitative+MGMT+promoter+methylation+analysis+using+pyrosequencing+and+combined+bisulfite+restriction+analysis.">Optimization of quantitative MGMT promoter methylation analysis using pyrosequencing and combined bisulfite restriction analysis.</a> The Journal of Molecular Diagnostics : JMD. 9 (3), 368-381 (2007).</li><li>Kreutz, M., Hochstein, N., Kaiser, J., Narz, F., Peist, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing:+powerful+and+quantitative+sequencing+technology.+Current+Protocols+In.">Pyrosequencing: powerful and quantitative sequencing technology. Current Protocols In.</a> Molecular Biology / edited by Frederick M. Ausubel ... [et al.]. 104 (Unit 7 15), (2013).</li><li>Dikow, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+methylation+status+of+the+PWS/AS+imprinted+region:+comparison+of+two+approaches+based+on+bisulfite+sequencing+and+methylation-sensitive+MLPA.">Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.</a> Molecular And Cellular Probes. 21 (3), 208-215 (2007).</li><li>Parrish, R. R., Day, J. J., Lubin, F. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+bisulfite+sequencing+for+examination+of+DNA+methylation+with+gene+and+nucleotide+resolution+from+brain+tissues.">Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues.</a> Current Protocols In Neuroscience / editorial board, Jacqueline N. Crawley ... [et al.]. 7 (Unit 7 24), (2012).</li><li>Shapiro, R., Servis, R. E., Welcher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactions+of+uracil+and+cytosine+derivatives+with+sodium+bisulfite.+A+specific+deamination+method.">Reactions of uracil and cytosine derivatives with sodium bisulfite. A specific deamination method.</a> Journal of the American Chemical Society. 92, 422-424 (1970).</li><li>Hayatsu, H., Wataya, Y., Kazushige, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+addition+of+sodium+bisulfite+to+uracil+and+to+cytosine.">The addition of sodium bisulfite to uracil and to cytosine.</a> Journal of the American Chemical Society. 92 (3), 724-726 (1970).</li><li>Wang, R. Y., Gehrke, C. W., Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+bisulfite+modification+of+5-methyldeoxycytidine+and+deoxycytidine+residues.">Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues.</a> Nucleic Acids Research. 8 (20), 4777-4790 (1980).</li><li>Masser, D. R., Berg, A. S., Focused Freeman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=high+accuracy+5-methylcytosine+quantitation+with+base+resolution+by+benchtop+next-generation+sequencing.">high accuracy 5-methylcytosine quantitation with base resolution by benchtop next-generation sequencing.</a> Epigenetics &amp; Chromatin. 6 (1), 33 (2013).</li><li>Caruccio, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+next-generation+sequencing+libraries+using+Nextera+technology:+simultaneous+DNA+fragmentation+and+adaptor+tagging+by+in+vitro+transposition.">Preparation of next-generation sequencing libraries using Nextera technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.</a> Methods in Molecular Biology. 733, 241-255 (2011).</li><li>Quail, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tale+of+three+next+generation+sequencing+platforms:+comparison+of+Ion+Torrent,+Pacific+Biosciences+and+Illumina+MiSeq+sequencers.">A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.</a> BMC Genomics. 13, 341 (2012).</li><li>Adey, A., Shendure, J. U. l. t. r. a. -. l. o. w. -. i. n. p. u. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tagmentation-based+whole-genome+bisulfite+sequencing.">tagmentation-based whole-genome bisulfite sequencing.</a> Genome Research. 22 (6), 1139-1143 (2012).</li><li>Smallwood, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genome-wide+bisulfite+sequencing+for+assessing+epigenetic+heterogeneity.">Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity.</a> Nature Methods. 11 (8), 817-820 (2014).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+endogenous+gene+controls+for+bisulphite+conversion+in+analysis+of+plant+DNA+methylation.">Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation.</a> Plant Methods. 7, 39 (2011).</li><li>Lister, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+DNA+methylomes+at+base+resolution+show+widespread+epigenomic+differences.">Human DNA methylomes at base resolution show widespread epigenomic differences.</a> Nature. 462 (7271), 315-322 (2009).</li><li>Ivanov, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-solution+hybrid+capture+of+bisulfite-converted+DNA+for+targeted+bisulfite+sequencing+of+174+ADME+genes.">In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes.</a> Nucleic Acids Research. 41 (6), e72 (2013).</li><li>Booth, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+bisulfite+sequencing+of+5-methylcytosine+and+5-hydroxymethylcytosine.">Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine.</a> Nature Protocols. 8 (10), 1841-1851 (2013).</li></ol>

The authors have nothing to disclose.

BSAS 샘플 및 대상의 수 두 수의 높은 처리량 기능에 초점을 맞춘, 정확한 DNA 메틸화 정량 수 있습니다. 또한, transposome 매개 tagmentation을 사용하여이 라이브러리 세대는 빠르고, 적은 입력 DNA입니다 필요로하며, 기존의 전단 및 후속 어댑터 결찰 기술보다 더 적은 단계가 포함되어 있습니다. 기존의 방법에 비해 이러한 개선은 관심의 대상의 정확한 기본 수준 DNA 메틸화 분석 할 수 있습니다. 또한, BSAS는 전체 게놈 중아 (22) 또는 캡처 (23) 접근 방식을 사용하여 식별 된 관심의 영역 (차별적 메틸화 지역 (DMRS)의 CpG 섬, 규제 지역)의 확인을위한 새로운 방법을 제공한다. 직교 확인 방법 등을 정량적으로 정확한 방법을 사용하여 에피 지노믹스 연구의 치밀한 분석을 향상시킨다. BSAS 달성 높은 읽기 깊이는 다시 좁은 신뢰 구간의 정확한 정량이 가능정확한 정량 16 sulting. 또한, 하나의 실험에서 많은 샘플을 실행할 수있는 능력은 통계적 전력을 증가 전체 게놈의 확정 방법에 대한 샘플 크기를 증가시킬 수있다; 많은 현재의 후생 유전 학적 연구의 약점. 중요한 것은, BSAS은 후생 유전 학적 연구에 대한 검증 가능한 가설의 생성을 허용베이스 고유의 CpG 메틸화 정량을 제공합니다. 이러한 특정 응답 요소의 증가 메틸화와 같은 가설이 감소 특정 전사 인자의 결합을 발생합니다. 한 쌍의 mRNA 발현 데이터와 결합 BSAS은 조직 또는 세포 집단의 단일 조각 내에 후생 유전 학적 규제 mRNA 발현 양을 얻기위한 방법을 제공한다. 규제와 표현의 짝 측정은 또한 과학적인 엄격함과 결과의 영향을 증가시킨다. BSAS 프로토콜 내에서 중요한 단계는 프라이머 디자인, 앰플 리콘 최적화 및 라이브러리 생성 / 포함QC. 프라이머가 적절하게 설계 효율이 다른 8로 증폭하지 않으면 PCR 바이어스가 도입된다. 이러한 효소 적으로 생성 된 100 % 및 0 % 시토신 메틸화 표준 같은 메틸화 표준의 사용은 어떤 경우 메틸화 정량 바이어스, 정도를 결정하는데 사용하고, 메틸화 (16)을 정량 할 때 적절한 방법론 제어하다 할 수있다. 단일 고품질의 증폭 물을 생성하기위한 PCR 반응을 최적화 할 때 마찬가지로주의를 기울여야한다. 앰플 리콘은 어떠한 프로토콜에서 상세히 제시된 지침을 이용하여 존재하지 않는 경우, 최적화 단계가 증가하는 PCR 사이클 번호 또는 bsDNA 입력량을 포함한다. 프라이머 이량 체 등 다양한 PCR 제품들이 존재하는 경우, 최적화 단계는, bsDNA 입력량 감소 PCR 프라이머 몰 입력 농도 저하, 소둔 온도의 증가, 또는 PCR 싸이클 횟수 감소 등. 이러한 최적화 절차 중 하나 또는 조합이 단일 HIG를 생성하기에 충분한 결과를 얻을H-품질 증폭 물. 또한, 프라이머를 재 설계하는 것은 하나의 증폭을 생성하지 않는 프라이머 세트를위한 좋은 방법입니다. 재 설계에 적합 또는 수 없습니다 그 지역의 경우 두 역 프라이머에서의 CpG 사이트에서의 CpG 앞으로 프라이머의 사이트와 아데닌의 구아닌의에서 시토신의 티민의이 작동을 포함 프라이머 세트를 퇴화. 그러나, 이들 프라이머 세트는 바이어스가 발생하는 어떠한 PCR,이 프로토콜의 범위를 벗어난 프로세스 없도록 위해 추가 최적화를 필요로한다. 품질 라이브러리는 성공을 위해 매우 중요합니다. 품질 관리 조치를 시퀀싱하기 전에 라이브러리의 크기, 질과 양을 결정하기 위해 수행되어 있는지 확인합니다. 시퀀싱 반응은 저품질 라이브러리의 입력에 실패하는 경향이있다. 메틸화 정량의 바이어스는 메틸화 주파수가 앞으로 모두에 존재하고 시퀀싱 읽기 역하도록함으로써 완화 될 수있다. 또한,의 CpG 사이트에 정렬 기지의 품질 평가 점수가 높은 협력을위한 ≥Q30해야정량에 nfidence. 다른 사람은 위에 설명 된 기준을 보장 중아 변환 된 DNA (19)의 tagmentation 반응에서 어떤 편견 이전에 작은 표시하고 있지만 낮은 바이어스 메틸화 정량의 확인을 할 수 있습니다. BSAS 현재 그러나,이 방법의 미래 확장은 바이 설 파이트 변환 24 전에 칼륨 퍼 루테 네이트를 도입으로하는 5- MC 및 5- HMC 구별 실험 단계에 첨가 된 CpG -5- HMC의 페어링 측정을 허용 소비재 부위에서 메틸화를 측정한다. 이것은 DNA 메틸화 유전자 발현 조절의 역할을 결정하기 위해, 한 쌍의 메틸화시킴으로써 5-MC 및 5- HMC 및 발현 데이터를 모두 BSAS 유틸리티의 영향을 증가시킬 것이다. PCR 증폭 산물의 Transposome 매개 라이브러리 제조 증폭 영역의 단부에서 감소 된 깊이 순서화 될 않는다. 따라서, 높은 관심의 영역이 증폭 산물의 중간에 위치하도록 설계되어야한다. 방법BSAS가 생성하기 때문에 지금까지, 시퀀싱&gt; 1000 X, 증폭 된 영역의 끝 근처 측정이 여전히 높은 5-MC의 양적 정도의 깊이를 읽어보십시오.

그것은 자연스럽게 시토신 메틸화 (1)의 형태로 DNA의 변형 발생의 첫 번째 보고서 이후 반세기에 걸쳐있다. 시토신 메틸화베이스 링에 5 번째 탄소 메틸기 (5-MC)는, 가장 빈번하게 포유 동물 게놈에서의 CpG 디 뉴클레오티드 모티브에서 발생 갖는 변성 시토신 뉴클레오티드 염기이다. 부재가 전사 활성과 연관이있는 동안 유전자 프로모터의 5-MC의 존재는 일반적으로 작용 성, 전사 억제와 관련된다. 엄청난 진보 발전 3의 DNA 메틸화, 후생 유전 학적 프로파일 4, 5,6 암 발병 기전 및 다른 연구 분야의 수의 transgeneration 전파의 역할에 대한 이해 만들어왔다. 신규 방법론은 DNA 메틸화 7 프로파일 개발되었다 이러한 많은 진보는 지난 몇 년 동안왔다. 출현 및차세대 시퀀싱 (NGS) 기술의 대중화는 이제 전체 게놈 또는 게놈 (8)의 큰 영역에 걸쳐 DNA 메틸화를 프로파일 링하는 방법은 여러 가지가있을 수 있습니다. 이러한 접근은 DNA 메틸화 패턴 및 DNA 메틸화의 차이를 정량화 연구의 발견 가능성을 연다. 이러한 가설 발생 방법에 더하여, 타겟 / 가설 테스트 DNA 메틸화 정량 방법에 대한 상당한 필요성이 존재한다. pyrosequencing 기법, 메틸화 특이 PCR, 바이 설 파이트 변환 된 DNA와 직접 생거 시퀀싱은 대상 영역의 분석을 위해 가장 많이 사용되는 방법이 있었다 (예를 들어, 하나의 유전자의 프로모터 영역 또는 CpG 아일랜드) 9-12. 메틸화 된 시토신의 그대로 13, 14을 유지하면서 이러한 모든 방법, 중아 변환, 메틸화 된 시토신의이 우라실의로 변환 화학 탈 아미 노화 반응에 의존하고있다. bisulfite-의 PCR 증폭차동 메틸화 염기 차이로서 판독 할 수 티민 15 우라실의 여분의 DNA에 변환 결과. 매우 유용하지만, 이러한 방법에 제한이 낮은 양적 정확성, 짧은 읽기 길이, 낮은 시료 처리를 포함한다. 이러한 한계를 해결하기 위해 우리는 우리가 (BSAS) 16 바이 설 파이트의 증폭 시퀀싱이라고 한 방법을 개발했다. 이 방법의 목적은 높은 정밀도로 정량 많은 수의 샘플에 대한 관심의 대상 게놈 영역을 분석 할 수 있어야한다. BSAS은 기존의 방법의 요소 (중아 변환 및 지역별 PCR 증폭)를 사용하고 간단한 차세대 라이브러리 건설 (transposome 매개) (17) 및 벤치 탑 NGS (18) (그림 1)로 결합되어 있습니다. 이 방법은 관심의 영역에서 시토신 메틸화를 검사 할 수있는 정량적 및 높은 처리량 방법을 제공한다. 여기에 우리가 제시세부 방법 및 문제 해결과 품질이 BSAS 프로세스를 확인하는 방법을 설명합니다.

<table><tbody><tr><td>Agilent 2100 Bioanlyzer</td><td>Agilent</td><td>G29393AA</td><td></td></tr><tr><td>Agilent RNA 6000 Nano kit</td><td>Agilent</td><td>5067-1511</td><td></td></tr><tr><td>Agilent High Sensitivity DNA kit</td><td>Agilent</td><td>5067-4626</td><td></td></tr><tr><td>Typhoon 9200</td><td>Amersham/GE Healthcare Life Sciences</td><td></td><td></td></tr><tr><td>Agencourt AMPure XP - PCR Purification</td><td>Beckman Coulter</td><td>A63880</td><td>5 ml</td></tr><tr><td>PowerPac Basic Power Supply</td><td>Bio Rad</td><td>164-5050</td><td></td></tr><tr><td>twin.tech PCR plate 96&amp;nbsp;</td><td>Eppendorf</td><td>951020362</td><td>semi-skirted</td></tr><tr><td>Heatsealing Film</td><td>Eppendorf</td><td>0030127838</td><td></td></tr><tr><td>Heatsealing Foil</td><td>Eppendorf</td><td>0030127854</td><td></td></tr><tr><td>Heat Sealer</td><td>Eppendorf</td><td>5390000.024</td><td></td></tr><tr><td>0.1-10 &amp;#956;l Tips</td><td>Eppendorf</td><td>0030073.002</td><td></td></tr><tr><td>2-200 &amp;#956;l Tips</td><td>Eppendorf</td><td>0030073.045</td><td></td></tr><tr><td>50-1,000 &amp;#956;l Tips</td><td>Eppendorf</td><td>0030073.100</td><td></td></tr><tr><td>MixMate&amp;nbsp;</td><td>Eppendorf</td><td>5353000.014</td><td></td></tr><tr><td>1.5 ml Microcentrifuge Tubes</td><td>Eppendorf</td><td>0030121.023</td><td></td></tr><tr><td>Centrifuge 5424</td><td>Eppendorf</td><td>5424000.010</td><td></td></tr><tr><td>2.0 ml Microcentrifuge Tube</td><td>Eppendorf</td><td>022363352</td><td></td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/products/nextera_xt_dna_library_prep_kit.html&quot;&gt;Nextera XT DNA Sample Preparation Kit&lt;/a&gt;</td><td>Illumina</td><td>FC-131-1024</td><td>24 rxn</td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/products/nextera_xt_dna_library_prep_kit.html&quot;&gt;Nextera XT Index Kit&lt;/a&gt;</td><td>Illumina</td><td>FC-131-1001</td><td>24 indexes</td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/systems/miseq.html&quot;&gt;MiSeq Desktop Sequencer&lt;/a&gt;</td><td>Illumina</td><td></td><td></td></tr><tr><td>&lt;a href=&quot;http://www.illumina.com/products/miseq_reagent_kit_v2.html&quot;&gt;MiSeq Reagent Kit v2&lt;/a&gt;</td><td>Illumina</td><td>MS-102-2002</td><td>300 cycles</td></tr><tr><td>KAPA SYBR FAST Universal qPCR kit</td><td>KAPA Biosystems</td><td>KK4824</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay kit</td><td>Life Technologies</td><td>P7589</td><td></td></tr><tr><td>Quant-iT RiboGreen RNA Assay kit</td><td>Life Technologies</td><td>R11490</td><td></td></tr><tr><td>ProFlex 96-well PCR system</td><td>Life Technologies</td><td>4484075</td><td></td></tr><tr><td>Novex 6% TBE DNA gel</td><td>Life Technologies</td><td>EC6261BOX</td><td>10-well</td></tr><tr><td>Novex TBE Running Buffer</td><td>Life Technologies</td><td>LC6675</td><td>5x (1 L)</td></tr><tr><td>Novex Hi-Density TBE Sample Buffer</td><td>Life Technologies</td><td>LC6678</td><td>5x (10 ml)</td></tr><tr><td>1 Kb Plus DNA Ladder</td><td>Life Technologies</td><td>10787-018</td><td></td></tr><tr><td>Xcell SureLock Mini-Cell</td><td>Life Technologies</td><td>EI0001</td><td></td></tr><tr><td>UltraPure 10 mg/ml Ethidium Bromide</td><td>Life Technologies</td><td>15585-011</td><td></td></tr><tr><td>7900HT Fast Real-Time PCR system</td><td>Life Technologies</td><td>4329001</td><td>384-well block</td></tr><tr><td>DynaMag-96 Side Skirted</td><td>Life Technologies</td><td>12027</td><td></td></tr><tr><td>SpectroMax M2 Multi-Mode Microplate Reader</td><td>Molecular Devices/VWR</td><td>89429-532</td><td></td></tr><tr><td>AllPrep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td></td></tr><tr><td>Stainless Steel Beads</td><td>Qiagen</td><td>69989</td><td>5 mm</td></tr><tr><td>CLC Genomics Workbench</td><td>Qiagen</td><td></td><td>Software for methylation pipeline</td></tr><tr><td>QIAQuick PCR Purification kit</td><td>Qiagen</td><td>28104</td><td>50 rxn</td></tr><tr><td>TissueLyser II</td><td>Retsch/Qiagen</td><td>85300</td><td></td></tr><tr><td>Nuclease-Free Water</td><td>Sigma</td><td>W4502</td><td></td></tr><tr><td>TRIS hydrochloride</td><td>Sigma</td><td>PHG0002-100G</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P9416-50ML</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>384-well Full-Skirted Plate, Standard</td><td>Thermo Scientific</td><td>AB-1384</td><td></td></tr><tr><td>Absolute qPCR Seal</td><td>Thermo Scientific</td><td>AB-1170</td><td></td></tr><tr><td>EZ DNA Methylation-Lightning kit</td><td>Zymo Research</td><td>D5030</td><td>50 rxn</td></tr><tr><td>ZymoTaq DNA Polymerase</td><td>Zymo Research</td><td>E2001</td><td>50 rxn</td></tr></tbody></table>

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.

BSAS 제대로도 5를 닮은 것 변환 된 레퍼런스 시퀀스에 정렬 읽는다. CpG 디 뉴클레오티드는 명확히 식별 할 수 있고, 메틸화 상태는 읽기에 매핑 된 CpG 부위에서베이스 호출을 관찰함으로써 추정 될 수있다. 예를 들어, 메틸화 컨트롤, 0 %는 모든 메틸화가 T의 (도 5a)를 함유 된 CpG 부위에 매핑 판독 될 것이다. 100 % 메틸화 컨트롤 매핑 읽기 모두가 C의 (그림 5B)를 포함 된 CpG 사이트에 대한 읽기가 발생합니다. 된 CpG 부위에서 주파수 시토신 (C / C + T)의 정량은 원래 샘플 메틸화 주파수를 산출한다. 전체 게놈의 메틸화 컨트롤 (N = 3 / 메틸화 비율)에서 발생하는 대표적인 표준 곡선은 각 메틸화 비율 (그림 6)에서 정량의 메틸화 정량의 선형성뿐만 아니라 정밀도를 보여줍니다. 총이 방법의 예로서, RNA 및 DNA는 공동 ISO이었다<html> 마우스 소뇌와 망막 (N = 4 / 그룹)에서 lated. 선택적 망막 조직에서 발현 로돕신 발현, qPCR을 사용하여 측정 하였다. 표현은 망막 (그림 7A)에서 검출되었다. BSAS를 사용하여, 메틸화 된 CpG 수준은 로돕신의 프로모터 영역에서 정량화 하였다. 프로모터 영역에 걸쳐 누적 메틸화 수준은 망막 (p &lt;0.001, 파라 메트릭 테스트 t) (도 7B)에서 &lt;15 %에 비해 소뇌에서&gt; 80 %이었다. 부위 특이 적 메틸화 정량로부터 BSAS 메틸화 정량 이점 및 메틸화 수준은 특정 지역 전체 게놈의 CpG 관련 기초 비교 될 수있다. 망막 샘플 (p &lt;0.001, 각 사이트에서의 CpG 파라 메트릭 테스트 t) (도 7C)에 비해 로돕신 프로모터 걸쳐 메틸화 된 CpG 소뇌 수준은 샘플에서 유의하게 높았다. 1highres.jpg &quot;/&gt; 그림 1 :. BSAS 방법의 도식이 방법에서는 게놈 DNA는 유라에 unmethylation의 사이토를 수정할 변환 중아입니다. 그 후, PCR 동안 이러한 유라는 thymines로 변경됩니다. PCR 증폭은 관심 영역에 대해 유도 및 높은 단지 이러한 시퀀스 풍부하게된다. 얻어진 PCR 증폭 산물은 간단한 공정을 통해 tagmentation 듀얼 인덱싱 라이브러리로 이루어진다. 그 후 라이브러리는 벤치 탑 차세대 시퀀서에 순서가되고, 순서는 시토신의는베이스 고유의 방식으로 결정된다 실리에서 변환 된 기준 서열과 퍼센트 메틸화에 매핑되는 읽습니다. <img alt="그림 2" src="/files/ftp_upload/52488/52488fig2highres.jpg" /> 그림 2 :. 관심 지역의 실리 중아 변환의 모든 게놈 기준에서 관심의 모든 영역이 SELEC 될 수 있습니다실리코 중아 변환에 대한 테드. 비의 CpG 사이토는 티민의 기준 순서로 대체됩니다. 의 CpG 사이토는 중아에서 사이토가 참조를 변환 남아있다. <img alt="그림 3" src="/files/ftp_upload/52488/52488fig3highres.jpg" /> 그림 3 :. 프라이머 배치 바이 설 파이트 PCR 프라이머가 실리 중아에 대해 설계는 레퍼런스 시퀀스로 변환. 뇌관은 CG 디 뉴클레오티드를 중첩하지 않도록 설계되거나 CG 디 뉴클레오티드에 인접 설계되어야한다. <img alt="그림 4" src="/files/ftp_upload/52488/52488fig4highres.jpg" /> 그림 4 : 높은과 낮은 품질의 PCR 증폭 산물과 염기 서열 라이브러리의 예 대표 electropherogram 추적 및 젤 쇼 (A) 하나 고와 transposome 매개 라이브러리 생성을위한 최적의 증폭.농도 제품입니다.이 증폭 물에서 생성 (B) 라이브러리 (C) 품질이 낮은 PCR 증폭 산물의 크기, 농도가 낮을 수있다. 고농도 심지어 크기 분포를 보여 주며, 여러 제품 및 / 또는 프라이머 이량 체를 포함한다. 이러한 낮은 품질의 증폭 산물은 (D)으로 이어질 낮은 농도와 낮은 크기 분포와 라이브러리 생성을 실패합니다. <img alt="그림 5" src="/files/ftp_upload/52488/52488fig5highres.jpg" /> 그림 5 : 메틸화 컨트롤에서 시퀀싱 출력의 예. 인 실리코 (A)가 된 CpG 사이트와 참조 순서가 빨간색으로 강조 변환. 매핑 0 % 메틸화 컨트롤의 CpG 사이트에서 선택한 영역 표시 thymines (탐욕 강조 표시) 매핑을 읽습니다. 매핑 (B) 100 % 메틸화 컨트롤의 CpG 사이트에서 시토신의 (파란색 강조 표시) 매핑을 보여 읽습니다. 도 6 :. 메틸화 컨트롤 범위에서 메틸화 정량 0 %에서 100 %까지 메틸화 비율 (N = 3 / 비)로 혼합 전체 게놈 메틸화 BSAS 컨트롤을 사용하여 정량 하였다. 정량화 대 예상의 정량을 플로팅 메틸화 제어 정량의 선형성을 보여줍니다. 모든 컨트롤에 걸쳐, 메틸화 정량의 높은 정밀도가 있었다. <img alt="그림 7" src="/files/ftp_upload/52488/52488fig7highres.jpg" /> 도 7 : 조직 샘플로부터의 페어링 된 DNA 메틸화와 유전자 발현 분석의 예. (A) 마우스 소뇌와 망막 조직에서 로돕신 상대 mRNA 발현. (B) 소뇌 및 망막 DNA (*** p &lt;0.001, 파라 메트릭 t-test를) 사이에 BSAS에 의해 정량화 로돕신 평균 프로모터 메틸화. &lt;마우스 소뇌와 망막 DNA의 전사 시작 사이트 [TSS])를 기준으로 로돕신 프로모터 (+6 -244에 걸쳐 강한&gt; C)의 CpG 사이트 별 메틸화 수준.

Watch this Scientific Journal Video about Targeted DNA Methylation Analysis by Next-generation Sequencing at JoVE.com

Targeted DNA Methylation Analysis by Next-generation 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 ...

targeted-dna-methylation-analysis-by-next-generation-sequencing

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Biology

37.0K 조회수.  University of Oklahoma College of Medicine. 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.

비디오: Targeted DNA Methylation Analysis by Next-generation Sequencing

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

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 ...

연구

유전학

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 ...

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 ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

쥐 메 틸-Seq 플랫폼 관련 스트레스에 노출 된 후 변경 내용을 확인 하

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

생화학

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

LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4&#8211;5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

Described here is the use of a methylation-specific probe amplification method to analyze methylation levels of LINE-1 elements in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles. Ultracentrifugation, a popular procedure for separating extracellular vesicles from fetal bovine serum, is also demonstrated.

골육종 유래 세포외 소포로 처리된 중간엽 줄기 세포의 LINE-1 메틸화 분석

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of ...

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

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA 메틸트랜스퍼라제 억제제를 스크리닝하기 위한 연속 형광 기반 엔도뉴클레아제 결합 DNA 메틸화 분석

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

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 ...

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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 ...