Name: bidirectional retroviral integration site pcr methodology and quantitative data analysis workflow
Uploaded: 2017-06-14T15:00:00
Description: Watch this Scientific Journal Video about Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow at JoVE.com

Finansiering blev ydet af National Institutes of Health Grants R00-HL116234, U19 AI117941 og R56 HL126544; National Science Foundation Grant DMS-1516675; Koreas Nationale Forskningsfond (NRF-2011-0030049, NRF-2014M3C9A3064552); Og KRIBB initiativprogrammet.

1. Generering af opstrøms (venstre) - og nedstrøms (højre) -junction sekvens biblioteker <ol><li> DNA linker præparation: <ol><li> Forbered en 10 μl linker-DNA-opløsning ved at tilsætte 2 μl 100 μM LINKER_A-oligos (endelige: 20 μM), 2 μl 100 μM LINKER_B-oligos (endelige: 20 μM), 2 μl 5 M NaCl (endelige: 1 M) og 4 μl nukleasefrit vand i et PCR-rør. Se tabel 1 for linkersekvenserne. </li><li> Inkubér linker-DNA-opløsningen ved 95 ° C i 5 minutter i et PCR-instrument, ...

<ol>
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Bio. 51 (1), 26-42 (2016).</li><li>Kim, S., 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=Dynamics+of+HSPC+Repopulation+in+Nonhuman+Primates+Revealed+by+a+Decade-Long+Clonal-Tracking+Study.">Dynamics of HSPC Repopulation in Nonhuman Primates Revealed by a Decade-Long Clonal-Tracking Study.</a> Cell Stem Cell. 14 (4), 473-485 (2014).</li><li>Bushman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+integration+and+human+gene+therapy.">Retroviral integration and human gene therapy.</a> J. Clin. Invest. 117 (8), 2083-2086 (2007).</li><li>Bystrykh, L. V., Verovskaya, E., Zwart, E., Broekhuis, M., de Haan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+stem+cells:+methodological+constraints.">Counting stem cells: methodological constraints.</a> Nat Meth. 9 (6), 567-574 (2012).</li><li>Schr&#246;der, 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=HIV-1+integration+in+the+human+genome+favors+active+genes+and+local+hotspots.">HIV-1 integration in the human genome favors active genes and local hotspots.</a> Cell. 110 (4), 521-529 (2002).</li><li>Silver, J., Keerikatte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+use+of+polymerase+chain+reaction+to+amplify+cellular+DNA+adjacent+to+an+integrated+provirus.">Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus.</a> J. 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Virol. 80 (22), 11313-11321 (2006).</li><li>Maldarelli, 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=Specific+HIV+integration+sites+are+linked+to+clonal+expansion+and+persistence+of+infected+cells.">Specific HIV integration sites are linked to clonal expansion and persistence of infected cells.</a> Science. 345 (6193), 179-183 (2014).</li><li>Wu, C., 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=High+Efficiency+Restriction+Enzyme-Free+Linear+Amplification-Mediated+Polymerase+Chain+Reaction+Approach+for+Tracking+Lentiviral+Integration+Sites+Does+Not+Abrogate+Retrieval+Bias.">High Efficiency Restriction Enzyme-Free Linear Amplification-Mediated Polymerase Chain Reaction Approach for Tracking Lentiviral Integration Sites Does Not Abrogate Retrieval Bias.</a> Hum. Gene. Ther. 24 (1), 38-47 (2013).</li><li>Aker, M., Tubb, J., Miller, D. G., Stamatoyannopoulos, G., Emery, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+Bias+of+Gammaretrovirus+Vectors+following+Transduction+and+Growth+of+Primary+Mouse+Hematopoietic+Progenitor+Cells+with+and+without+Selection.">Integration Bias of Gammaretrovirus Vectors following Transduction and Growth of Primary Mouse Hematopoietic Progenitor Cells with and without Selection.</a> Mol. Ther. 14 (2), 226-235 (2006).</li><li>Gabriel, R., Kutschera, I., Bartholomae, C. C., von Kalle, C., Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+Amplification+Mediated+PCR;+Localization+of+Genetic+Elements+and+Characterization+of+Unknown+Flanking+DNA.">Linear Amplification Mediated PCR; Localization of Genetic Elements and Characterization of Unknown Flanking DNA.</a> J Vis Exp. (88), e51543 (2014).</li><li>Kim, S., 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=High-throughput,+sensitive+quantification+of+repopulating+hematopoietic+stem+cell+clones.">High-throughput, sensitive quantification of repopulating hematopoietic stem cell clones.</a> J. Virol. 84 (22), 11771-11780 (2010).</li><li>Mitchell, 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=Retroviral+DNA+integration:+ASLV,+HIV,+and+MLV+show+distinct+target+site+preferences.">Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.</a> PLoS Biol. 2 (8), e234 (2004).</li><li>Melkus, 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=Humanized+mice+mount+specific+adaptive+and+innate+immune+responses+to+EBV+and+TSST-1.">Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.</a> Nat. Med. 12 (11), 1316-1322 (2006).</li><li>Bystrykh, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combinatorial+approach+to+the+restriction+of+a+mouse+genome.">A combinatorial approach to the restriction of a mouse genome.</a> BMC Res Notes. 6, 284 (2013).</li><li>Beard, B. C., Adair, J. E., Trobridge, G. D., Kiem, H. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genomic+mapping+of+vector+integration+sites+in+gene+therapy+studies.">High-throughput genomic mapping of vector integration sites in gene therapy studies.</a> Methods Mol Biol. , 321-344 (2014).</li><li>Gabriel, 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=Comprehensive+genomic+access+to+vector+integration+in+clinical+gene+therapy.">Comprehensive genomic access to vector integration in clinical gene therapy.</a> Nat. Med. 15 (12), 1431-1436 (2009).</li><li>Qin, X., An, D., Chen, I., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+HIV-1+infection+in+human+T+cells+by+lentiviral-mediated+delivery+of+small+interfering+RNA+against+CCR5.">Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5.</a> Proc Natl Acad Sci U S A. 100 (1), 183-188 (2003).</li><li>Kitamura, 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=Retrovirus-mediated+gene+transfer+and+expression+cloning:+powerful+tools+in+functional+genomics.">Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics.</a> Exp. Hematol. 31 (11), 1007-1014 (2003).</li><li>Cartier, 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=Hematopoietic+stem+cell+gene+therapy+with+a+lentiviral+vector+in+X-linked+adrenoleukodystrophy.">Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.</a> Science. 326 (5954), 818-823 (2009).</li></ol>

Det tovejsede assay muliggør samtidig analyse af både opstrøms (venstre) og nedstrøms (højre) vektor-vært-DNA-forbindelsessekvenser og er anvendelig i en række genterapi-, stamceller- og cancerforskningsapplikationer. Anvendelsen af ​​GTAC-motiv-enzymer (RsaI og CviQI) og den tovejsede PCR-tilgang forbedrer signifikant chancerne for at detektere en integromet (eller en klonal population) sammenlignet med tidligere TCGA-motiv enzym ( TaqaI ) -baserede assays 2 ,</...

Retrovirus indsætter deres genomiske DNA i værtsgenomet på forskellige steder. Denne unikke egenskab, som kan bidrage til udviklingen af ​​kræftformer og andre former for viral patogenese, har den ironiske fordel at gøre disse vira yderst velegnede til celleteknik til genterapi og grundbiologiforskning. Viral Integration Site (IS) - placeringen på værtsgenomet hvor et fremmed DNA (virus) er integreret - har vigtige implikationer for skæbnen for både de integrerede vira og værtscellerne. IS-analyser er blevet anvendt i forskellige biologiske og kliniske forskningsinstitutioner for at studere retroviral integrationssiteudvælgelse og patogenese, kræftudvikling, stamcellebiologi og udviklingsbiologi 1 , 2 , 3 , 4 . Lav detektionsfølsomhed, dårlig data reproducerbarhed og hyppig krydskontaminering er blandtNøglefaktorerne begrænser anvendelsen af ​​IS-analyser til aktuelle og planlagte undersøgelser. Mange IS-analyseteknologier er blevet udviklet. Restriktionsenzymbaserede integrationssite-assays, herunder linker-medieret (LM) polymerase-kædereaktion (PCR) 5 , inverse PCR 6 og lineærforstærkningsmedieret (LAM) PCR 7 er de mest anvendte. Anvendelsen af ​​stedsspecifikke restriktionsenzymer genererer imidlertid en bias under hentningen af ​​IS, hvilket tillader kun en delmængde af integromer (et fremmed DNA integreret i værtsgenomet) i nærheden af ​​restriktionsstedet, der skal genvindes 4 . Analyseteknologier, der mere omfattende vurderer vektor IS, er også blevet introduceret i de seneste år. Disse analyser anvender forskellige strategier, herunder Mu transposon-medieret PCR 8 , ikke-restriktive (nr) -LAM PCR 9 , type II restriktionsenzym-mEdieret fordøjelse 10 , mekanisk forskydning 11 og tilfældig hexamerbaseret PCR (Re-fri PCR) 12 til fragmentering af genomiske DNA&#39;er og amplificering af IS. Nuværende teknologier har varierende niveauer af detektionsfølsomhed, genomdækning, målspecificitet, høj kapacitetskapacitet, kompleksitet af analyseprocedurer og forstyrrelser ved detektering af de relative frekvenser af målsteder. I betragtning af de forskellige kvaliteter af de eksisterende analyser og de forskellige formål, som de kan anvendes til, bør den optimale analysemetode udvælges omhyggeligt. Dette arbejde giver detaljerede eksperimentelle procedurer og en beregningsdataanalyseproces for et tovejsanalyse, der signifikant forbedrer detektionshastigheder og sekvenskvantificeringsnøjagtighed ved samtidigt at analysere IS opstrøms og nedstrøms for det integrerede mål-DNA (se figur 1 for et skematisk billede af analyseprocedurerne ). Denne tilgang giver ogsåS midler til at karakterisere den retrovirale integrationsproces (for eksempel troværdigheden af ​​målstedduplikation og variationer i de genomiske sekvenser af opstrøms og nedstrøms insertioner). Andre bidirektionelle metoder er primært brugt til kloning og sekventering af begge ender af mål-DNA&#39;et 11 , 13 , 14 . Dette assay optimeres i høj grad for den høje gennemstrømning og reproducerbare kvantificering af vektormarkerede kloner ved anvendelse af den veletablerede LM-PCR-metode og beregningsanalyse-kortlægning og til kvantificering af både opstrøms og nedstrøms krydsninger. Tovejsanalyse med TaqaI- enzymet har vist sig nyttigt til højkvalitets klonal kvantificering i stamcelle genterapi prækliniske studier 2 , 15 . Dette papir beskriver en ændret metode ved hjælp af en hyppigere cutter (RsaI / CviQI - motiv: GTAC), der fordobler tHan chancer for at detektere integromer sammenlignet med et TaqaI- baseret assay . Detaljerede forsøgs- og dataanalyseprocedurer, der bruger GTAC-motiv enzymer til lentiviral (NL4.3 og dets derivater) og gamma-retroviral (pMX vektorer) vektor IS-analyse, er beskrevet. Oligonukleotiderne anvendt i assayet er anført i tabel 1 . Et internt programmeringsskript til IS-sekvensanalyse findes i supplerende dokument.

<table border="0" cellpadding="0" cellspacing="0" width="1004">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Thermostable DNA polymerase</td>
			<td style="width:208px;">Agilent</td>
			<td style="width:119px;">600424</td>
			<td style="width:347px;">PicoMaxx Polymerase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermostable DNA polymerase buffer</td>
			<td style="width:208px;">Agilent</td>
			<td>600424</td>
			<td style="width:347px;">PicoMaxx Polymerase buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Deoxynucleotide (dNTP) solution mix</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>N0447L</td>
			<td>dNTP solution mix (10mM each)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR tubes</td>
			<td style="width:208px;">VWR International</td>
			<td style="width:119px;">53509-304</td>
			<td style="width:347px;">PCR Strip Tubes With Individual Attached Caps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">2ml microcentrifuge tube</td>
			<td style="width:208px;">Molecular Bioproducts</td>
			<td style="width:119px;">3453</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">PCR purification kit</td>
			<td style="width:208px;">Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">RsaI&#160;</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>R0167L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">CviQI</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>R0639L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Buffer A</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>B7204S</td>
			<td>NEB CutSmart buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Polymerase I large (klenow) fragment&#160;</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>M0210L</td>
			<td>Blunting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptavidin beads solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Binding Solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Washing Solution</td>
			<td>Invitrogen&#160;</td>
			<td>60101</td>
			<td>Dynabeads kilobaseBINDER kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic stand</td>
			<td style="width:208px;">ThermoFisher</td>
			<td>12321D</td>
			<td>DynaMag&#8482;-2 Magnet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">T4 DNA ligase</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>M0202L</td>
			<td>T4 DNA ligase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">10X T4 DNA ligase buffer</td>
			<td style="width:208px;">New England Biolabs</td>
			<td>B0202S</td>
			<td>T4 DNA ligase reaction buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">5X T4 DNA ligase buffer</td>
			<td style="width:208px;">Invitrogen&#160;</td>
			<td>46300-018</td>
			<td>T4 DNA ligase buffer with polyethylene glycol-8000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-Vis spectrophotometer</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>S06497</td>
			<td style="width:347px;">Nanodrop 2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pvuII</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>R0151L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">sfoI</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>R0606L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buffer B</td>
			<td style="width:208px;">new England Biolabs</td>
			<td>B7203S</td>
			<td>NEB buffer 3.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nuclease free water</td>
			<td style="width:208px;">Integrated DNA Technologies</td>
			<td>11-05-01-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Capillary electrophoresis</td>
			<td style="width:208px;">Qiagen</td>
			<td>9001941</td>
			<td style="width:347px;">QIAxcel capillary electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Veriti 96-well Fast Thermal Cycler</td>
			<td style="width:208px;">Thermo Fisher Scientific</td>
			<td>4375305</td>
			<td>PCR Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Rotating wheel (or Roller)</td>
			<td style="width:208px;">Eppendorf</td>
			<td>M10534004</td>
			<td>Cell Culture Roller Drums</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA size marker</td>
			<td style="width:208px;">Qiagen</td>
			<td>929559</td>
			<td>QX size marker (100-2,500 bp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA size marker</td>
			<td style="width:208px;">Qiagen</td>
			<td>929554</td>
			<td>QX size marker (50-1,500 bp)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">DNA alignment markers</td>
			<td style="width:208px;">Qiagen</td>
			<td>929524</td>
			<td>QX DNA Alignment Marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">genomc DNA</td>
			<td style="width:208px;">Not Available</td>
			<td style="width:119px;">Not Available</td>
			<td>Sample genomc DNA from in vivo or in vitro experiments</td>
		</tr>
	</tbody>
</table>

bidirectional retroviral integration site pcr methodology and quantitative data analysis workflow

Integration Site (IS) analyser er en kritisk komponent i studiet af retrovirale integrationssteder og deres biologiske betydning. I nyere retrovirale genterapiundersøgelser er IS-analyser i kombination med næste generations sekventering blevet anvendt som et celleopsparingsværktøj til karakterisering af klonale stamcellepopulationer, der deler samme IS. For den nøjagtige sammenligning af repopulerende stamcellekloner inden for og på tværs af forskellige prøver er detektionens følsomhed, data reproducerbarhed og høj gennemløbskapacitet af analysen blandt de vigtigste analysekvaliteter. Dette arbejde giver en detaljeret protokol- og dataanalyseproces for tovejs-IS-analyse. Det tovejsede assay kan samtidigt sekvensere både opstrøms og nedstrøms vektor-værtsforbindelser. Sammenlignet med konventionelle ensrettede IS-sekventeringsmetoder forbedrer den tovejstilgang signifikant IS-detektionshastigheder og karakteriseringen af ​​integrationshændelser i begge ender af tHan målretter DNA. Dataanalysepipelinjen beskrevet her identificerer og opregner identiske og identiske identiske sekvenser gennem flere trin af sammenligning, som kort IS sekvenser på referencegenomet og bestemmer sekventeringsfejl. Ved hjælp af en optimeret analyseprocedure har vi for nylig offentliggjort de detaljerede repopulation mønstre af tusinder af hæmatopoietiske stamceller (HSC) kloner efter transplantation i rhesus macaques, der for første gang demonstrerer det præcise tidspunkt for HSC-repopulation og den funktionelle heterogenitet af HSC&#39;er i Primatsystem. Den følgende protokol beskriver trin for trin eksperimentel procedure og dataanalyse-arbejdsgang, der identificerer og kvantificerer identiske IS-sekvenser nøjagtigt.

Den tovejsede IS-analyse genererede forskellige størrelser af PCR-amplikoner for både opstrøms (venstre) og nedstrøms (højre) vektorværtsforbindelser ( figur 2 ). Størrelsen af ​​en PCR-amplicon er afhængig af placeringen af ​​det nærmeste GTAC-motiv opstrøms og nedstrøms for en integrering. Assayet producerede også interne DNA-PCR-amplikoner: retrovirale sekvenser nær polypurinkanalen og primerbindingsstedet blev samtidig amplificeret u...

Watch this Scientific Journal Video about Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow at JoVE.com

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Dette manuskript beskriver den eksperimentelle procedure og softwareanalyse for et tovejs integrationssite-assay, der samtidigt kan analysere opstrøms og nedstrøms vektor-værtsforbindelses-DNA. Tovejsede PCR-produkter kan anvendes til enhver downstream-sekventeringsplatform. De resulterende data er nyttige til en høj-gennemløbs kvantitativ sammenligning af integrerede DNA-mål.

Tovejs Retroviral Integration Site PCR Metode og Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Research

JoVE Journal

Genetics

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Fletning absolutte og relative kvantitativ PCR data at kvantificere STAT3 Splice Variant Afskrifter

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

Påvisning af mikroRNA ekspression i peritoneal membran af rotter ved hjælp af kvantitativ real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Retroviral Scanning: Kortlægning af MLV Integration Sites til at definere Cell-specifikke Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

Forberedelse af prøver og analyser af RNASeq-baseret genekspression Data fra zebrafisk

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

En standardmetode til at undersøge ejendommen mutagenicitet som en funktion af punkt Mutation reparation katalyseret af CRISPR/Cas9 og SsODN i humane celler

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Metoder til nøjagtig detektering af mitokondrie-DNA methylering

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

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

IT analyse af sekvens Data fra Batch gær 2-Hybrid skærme

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Multi-locus variabel-tal tandem-Gentag analyse af den fisk-patogene bakterie Yersinia ruckeri ved MULTIPLEX PCR og kapillar elektroforese

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digitalt PCR-baseret konkurrencemæssigt indeks til analyse af egnethed til salmonella med høj gennemløb

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

Kvantitative RT-PCR'er med høj gennemløb i enkelt- og bulk-C. elegans-prøver ved hjælp af nanofluidisk teknologi

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...