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

Arbejdet er støttet af CPRIT Research Training Award RP140105, samt delvist støttet af amerikanske National Institutes of Health (NIH) giver R01 GM114142 og af William &amp; Ella Owens Medical Research Foundation.

Alle væv opnås efter godkendelse af Institutional Review Board udvalg og når alle deltagere samtykke til både molekylære analyser og opfølgende undersøgelser. Protokollerne er godkendt af udvalget Menneskelige Studies ved University of Texas Health Science Center på San Antonio. 1. Methyl-bindende DNA Capture (MBDCap) <ol><li> Indsamling prøve og DNA-isolering <ol><li> Indsamle bulk-tumor eller normale vævsprøver fra patientens paraffinindlejrede vævsprøver. </li><li> Brug en komm...

<ol>
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L., 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=DNA+methylation+profiling:+comparison+of+genome-wide+sequencing+methods+and+the+Infinium+Human+Methylation+450+Bead+Chip.">DNA methylation profiling: comparison of genome-wide sequencing methods and the Infinium Human Methylation 450 Bead Chip.</a> Epigenomics. , 1-16 (2015).</li><li>Huang, Y. W., Huang, T. H., Wang, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+DNA+methylomes+from+microarray+to+genome-scale+sequencing.">Profiling DNA methylomes from microarray to genome-scale sequencing.</a> Technol. Cancer Res. Treat. 9, 139-147 (2010).</li><li>Serre, D., Lee, B. H., Ting, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MBD-isolated+Genome+Sequencing+provides+a+high-throughput+and+comprehensive+survey+of+DNA+methylation+in+the+human+genome.">MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome.</a> Nucleic Acids Res. 38, 391-399 (2010).</li><li>Brinkman, A. B., 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=Whole-genome+DNA+methylation+profiling+using+MethylCap-seq.">Whole-genome DNA methylation profiling using MethylCap-seq.</a> Methods. 52, 232-236 (2010).</li><li>Wang, 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=LOcating+non-unique+matched+tags+(LONUT)+to+improve+the+detection+of+the+enriched+regions+for+ChIP-seq+data.">LOcating non-unique matched tags (LONUT) to improve the detection of the enriched regions for ChIP-seq data.</a> PLoS One. 8, e67788 (2013).</li><li>Gu, F., 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=CMS:+a+web-based+system+for+visualization+and+analysis+of+genome-wide+methylation+data+of+human+cancers.">CMS: a web-based system for visualization and analysis of genome-wide methylation data of human cancers.</a> PLoS One. 8, e60980 (2013).</li><li>Lan, X., Bonneville, R., Apostolos, J., Wu, W., Jin, V. 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Den MBDCap-seq teknik er en affinitet berigelse tilgang 3, betragtes som et omkostningseffektivt alternativ når han undersøger kohorter med et stort antal patienter 15. Rørledningen præsenteres her beskriver en samlet tilgang fra prøve indkøb til analyse og fortolkning af data. Et af de vigtigste skridt er at oprette en PCR-amplifikation for at sikre bedre PCR effektivitet GC beriget regioner i genomet som dette er hvor DNA-methylering forekommer. Det er også vigtigt at sikre, at efter sekventering, hver prøve ...

Epigenetisk regulering af gener gennem DNA-methylering er en af de væsentlige mekanismer, der kræves til bestemmelse af celle skæbne ved stabil differentiering af forskellige vævstyper i legemet 1. Dysregulering af denne proces er blevet kendt for at forårsage forskellige sygdomme, herunder cancer 2. Denne fremgangsmåde involverer hovedsagelig tilsætning af methylgrupper på cytosinrest i CpG-dinukleotider i DNA 3. Der er et par forskellige teknikker i øjeblikket anvendes til at undersøge denne mekanisme, der hver har deres egne fordele som skitseret i mange undersøgelser 2-8. Her vil vi diskutere en af ​​disse teknikker kaldes Methyl-bindende DNA Capture sekventering (MBDCap-seq), hvor vi bruger en affinitet berigelse teknik til at identificere methylerede regioner af DNA. Denne teknik bygger på den methyl-bindende evne MBD2 proteinet til berige for de genomiske DNA-fragmenter indeholdende methylerede CpG sites. Vi benytter en kommerciel methyleret DNA berigelse kittil isoleringen af ​​disse methylerede regioner. Vores laboratorium har screenet hundredvis af patientprøver ved hjælp af denne teknik, og her giver vi et omfattende optimeret protokol, som kan bruges til at undersøge store patientkohorter. Som det ses med enhver næste generation sekventering teknologi, MBDCap-seq kræver også en specifikke bioinformatik tilgang for nøjagtigt at kvantificere niveauet af methylering tværs prøverne. Der har været mange nylige undersøgelser i et forsøg på at optimere normalisering og analyseprocessen af sekventering data 9, 10. I denne protokol, vi demonstrerer en af ​​disse metoder til gennemførelse af en unik læse opsving tilgang - LONUT - efterfulgt af lineær normalisering af hver prøve for at muliggøre upartiske sammenligninger på tværs stort antal patientprøver.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>3M sodium acetate pH 5.2</td>
			<td>Sigma</td>
			<td>S7899</td>
			<td>100ml</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

Methylering er en af ​​de væsentlige epigenetiske ændringer af DNA, som er ansvarlig for den præcise regulering af gener, der er nødvendige for stabil udvikling og differentiering af forskellige vævstyper. Dysregulering af denne proces er ofte kendetegnende for forskellige sygdomme som kræft. Her vil vi skitsere en af ​​de seneste sekventering teknikker, Methyl-bindende DNA Capture sekventering (MBDCap-seq), anvendes til at kvantificere methylering i forskellige normale og sygdom væv til store patientkohorter. Vi beskriver en detaljeret protokol af denne affinitetsberigelse tilgang sammen med en bioinformatik rørledning for at opnå optimal kvantificering. Denne teknik er blevet anvendt til at sekventere flere hundrede patienter på tværs af forskellige cancertyper som en del af det 1.000 methylome projektet (Cancer Methylome System).

Vi har anvendt MBDCap-seq at studere DNA methylering ændringer i et stort antal patienter fra forskellige cancertyper, herunder bryst- 12, endometrial 13, prostata 14 og leverkræft blandt andre. Her demonstrerer vi nogle oplysninger fra undersøgelsen brystkræft offentliggjort for nylig 12. I dette tilfælde benyttede vi hele genomet sekventering tilgang til at identificere CpG øer, der er differentielt methylerede i tumor i forhold til normalt tværs af forskellige genomiske regioner. Unders?...

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

Her præsenterer vi en protokol til at undersøge genomet bred DNA methylering i stor skala kliniske patient screening undersøgelser med anvendelse af methyl-Bindende DNA Capture sekventering (MBDCap-seq eller MBD-seq) teknologi og efterfølgende bioinformatik analyse pipeline.

Methyl-bindende DNA capture sekventering for Patient Væv

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

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