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 tillhandahölls av National Institutes of Health Grants R00-HL116234, U19 AI117941 och R56 HL126544; National Science Foundation Grant DMS-1516675; Koreas nationella forskningsstiftelse (NRF-2011-0030049, NRF-2014M3C9A3064552); Och KRIBB-initiativprogrammet.

1. Generera uppströms (vänster) - och nedströms (höger) -junktionssekvensbiblioteken <ol><li> DNA-linkerpreparat: <ol><li> Förbered en 10 μl linker-DNA-lösning genom att tillsätta 2 μl 100 μM LINKER_A-oligos (slutlig: 20 μM), 2 μl 100 μM LINKER_B-oligos (slutlig: 20 μM), 2 μl 5 M NaCl (slutlig: 1 M) och 4 pl nukleasfritt vatten i ett PCR-rör. Se tabell 1 för länksekvenserna. </li><li> Inkubera linker-DNA-lösningen vid 95 ° C under 5 minuter i ett PCR-instrument, st...

<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. 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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>

Tvåriktningsanalysen möjliggör samtidig analys av både uppströms (vänster) och nedströms (höger) vektor-värd-DNA-förbindningssekvenser och är användbar i ett antal genterapi-, stamceller- och cancerforskningsapplikationer. Användningen av GTAC-motiv-enzymer (RsaI och CviQI) och det dubbelriktade PCR-förfarandet förbättrar signifikant chanserna för detektering av en integrom (eller en klonal population) jämfört med tidigare TCGA-motiv-enzym ( Taqal ) -baserade analyser <sup class="xre...

Retrovirus sätter in deras genomiska DNA i värdgenomet på olika ställen. Denna unika egenskap, som kan bidra till utvecklingen av cancer och andra former av viral patogenes, har en ironisk fördel att göra dessa virus mycket mottagliga för cellteknik för genterapi och grundbiologiforskning. Viral Integration Site (IS) - placeringen på värdgenomet där ett främmande DNA (virus) integreras - har viktiga konsekvenser för ödet för både de integrerade virusen och värdcellerna. IS-analyser har använts i olika biologiska och kliniska forskningsinställningar för att studera retroviralt integrationsställsval och patogenes, cancerutveckling, stamcellsbiologi och utvecklingsbiologi 1 , 2 , 3 , 4 . Låg detekteringskänslighet, dålig datareproducerbarhet och frekvent korsförorening är blandNyckelfaktorerna begränsar användningen av IS-analyser till aktuella och planerade studier. Många IS-analystekniker har utvecklats. Restriktionsenzymbaserade integrationsstadsanalyser, inklusive Linker-medierad (LM) polymerasekedjereaktion (PCR) 5 , invers PCR 6 och linjärförstärkningsmedierad (LAM) PCR 7 är de mest använda. Användningen av platsspecifika restriktionsenzymer genererar emellertid en bias vid återhämtningen av IS, vilket möjliggör endast en delmängd integrerade (ett främmande DNA integrerat i värdgenomet) i närheten av restriktionsstället som skall återvinnas 4 . Analysteknik som mer omfattande utvärderar vektor IS har också introducerats de senaste åren. Dessa analyser använder olika strategier, innefattande Mu transposon-medierad PCR 8 , icke-restriktiv (nr) -LAM PCR 9 , typ II-restriktionsenzym-mEdierad digestion 10 , mekanisk skjuvning 11 och slumpmässig hexamerbaserad PCR (Re-free PCR) 12 , för fragmentering av genomiska DNA och amplifiering av IS. Nuvarande tekniker har varierande nivåer av detekteringskänslighet, genomdäckning, målspecificitet, hög genomströmningskapacitet, analysprocedurernas komplexitet och förspänningar vid detektering av de relativa frekvenserna hos målplatserna. Med tanke på de olika egenskaperna hos de befintliga analyserna och de olika syften för vilka de kan användas, bör den optimala analysmetoden noggrant väljas. Detta arbete tillhandahåller detaljerade experimentella förfaranden och ett arbetsdata för databasanalys för en dubbelriktad analys som avsevärt förbättrar detekteringshastigheten och sekvenskvantifieringsnoggrannheten genom att samtidigt analysera IS uppströms och nedströms om det integrerade mål-DNA (se Figur 1 för en schematisk bild av analysprocedurerna ). Detta tillvägagångssätt ger ocksåS medel för att karakterisera den retrovirala integrationsprocessen (till exempel trovärdigheten för målplats duplicering och variationer i de genomiska sekvenserna av uppströms och nedströms införingar). Andra bidirektionsmetoder har använts huvudsakligen för kloning och sekvensering av båda ändarna av mål-DNA 11 , 13 , 14 . Denna analys optimeras i stor utsträckning för hög genomströmning och reproducerbar kvantifiering av vektormärkta kloner, med användning av den väletablerade LM-PCR-metoden och kartläggning av analysanalys och för kvantifiering av både uppströms och nedströms förbindelser. Tvåriktningsanalys med TaqaI- enzymet har visat sig vara användbart för högklassig klonal kvantifiering i prekliniska studier av stamcellsgeneration 2 , 15 . I detta dokument beskrivs en modifierad metod med en frekventare skärare (RsaI / CviQI - motiv: GTAC) som dubblerar tHan chanser att detektera integrer jämfört med en Taqal- baserad analys . Detaljerade experimentella och data analysmetoder som använder GTAC-motiv enzymer för lentivirala (NL4.3 och dess derivat) och gamma-retrovirala (pMX vektorer) vektor IS-analys beskrivs. De oligonukleotider som användes vid analysen är listade i tabell 1 . Ett internt programmeringsskript för IS-sekvensanalys finns i kompletteringsdokumentet.

<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 är en kritisk komponent i studien av retrovirala integrationsställen och deras biologiska betydelse. I de senaste retrovirala genterapistudierna har IS-analyser, i kombination med nästa generationens sekvensering, använts som ett cellspårningsverktyg för att karakterisera klonala stamcellspopulationer som delar samma IS. För den korrekta jämförelsen av repopulerande stamcellkloner inom och över olika prover är detektionens känslighet, dataframtagbarhet och hög genomströmningskapacitet hos analysen bland de viktigaste analysegenskaperna. Det här arbetet ger ett detaljerat protokoll- och dataanalys-arbetsflöde för dubbelriktad IS-analys. Tvåriktningsanalysen kan samtidigt sekvensera både uppströms och nedströms vektor-värdkryssningar. Jämfört med konventionella enriktade IS-sekvenseringsmetoder, förbättrar dubbelriktat tillvägagångssätt signifikant IS-detekteringshastigheter och karakteriseringen av integrationshändelser i båda ändarna av tHan riktar sig mot DNA. Dataanalyspipelinjen som beskrivs här identifierar och summerar identiska IS-sekvenser genom flera steg för jämförelse som kartlägger IS-sekvenser på referensgenomet och bestämmer sekvenseringsfel. Med hjälp av ett optimerat analysförfarande har vi nyligen publicerat detaljerade återuppbyggnadsmönster av tusentals hematopoetiska stamceller (HSC) efter transplantation i rhesusmakar, vilket för första gången demonstrerar den exakta tidpunkten för HSC-repopulationen och den funktionella heterogeniteten hos HSCs i Primatsystem. Följande protokoll beskriver steg för steg experimentell procedur och dataanalys arbetsflöde som exakt identifierar och kvantifierar identiska IS-sekvenser.

Den tvåriktiga IS-analysen genererade olika storlekar av PCR-ampliconer för både uppströms (vänster) och nedströms (höger) vektorvärdesövergångar ( Figur 2 ). Storleken på ett PCR-amplicon är beroende av placeringen av närmaste GTAC-motiv uppströms och nedströms från en integrometod. Analysen producerade också interna DNA-PCR-amplikoner: retrovirala sekvenser nära polypurinområdet och primerbindningsstället förstärktes samtidigt under v...

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

Detta manuskript beskriver experimentell procedur och mjukvaruanalys för en dubbelriktad integreringsställeanalys som samtidigt kan analysera uppströms och nedströms vektor-värd-kryssnings-DNA. Tvåriktiga PCR-produkter kan användas för någon nedströms sekvenseringsplattform. Den resulterande data är användbar för en hög genomströmning, kvantitativ jämförelse av integrerade DNA-mål.

Tvåriktad retroviral integrationsplats PCR-metodik och kvantitativ dataanalys arbetsflöde

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

The overall goal of this procedure is to identify retroviral vector integration sites in the host genome and quantify the relative frequencies of clonal cell populations each sharing the same integration site. This method can help answer key questions in retroviral gene therapy, such as which part of the host genome vector has integrated, how a genetically engineered cell will repopulate after the transplant, and how they are functionally different. The main advantage of this technique is that retro-integration site sequences can be analyzed with higher accuracy and sensitivity by sequencing both upstream and downstream vector host DNA junctions simultaneously.Though this method can provide insight into the safety of vectors and gene engineered cell in gene therapy studies, it can also be applied to other basic biology studies to investigate individual cell behavior in vivo. The first step in this procedure is to determine the DNA concentration and the ratio of absorbance at 260 and 280 nanometers of the genomic DNA to be analyzed. After this, dilute one to two micrograms of the sample genomic DNA to a final volume of 170 microliters of genomic DNA solution using nuclease free water.Prepare a 200 microliter PCR reaction by mixing the following in a microcentrifuge tube. 2.5 microliters each of HIV-1 specific biotin primers, or a total of 10 microliters for four lentivirus-specific primers, 20 microliters of 10X thermostable DNA polymerase buffer, four microliters of 10 millimolar dNTPs, three microliters of thermostable DNA polymerase, and 163 microliters of genomic DNA. Divide the solution equally into four PCR tubes and carry out a single extension cycle under the following condition.94 degrees Celsius for 5 minutes, 56 degrees Celsius for 3 minutes, 72 degrees Celsius for 5 minutes, and 4 degrees C for storage. Pool all four PCR reactions into a two milliliter microcentrifuge tube. Follow the manufacturer's procedure of a PCR purification kit, and elute with 50 microliters of elution buffer.Prepare for a 100 microliter digestion by adding the following to a microcentrifuge tube. 50 microliters of DNA, 10 microliters of 10X buffer A, two microliters of RSAI enzyme, and 38 microliters of nuclease-free water. Incubate at 37 degrees Celsius in a thermal cycler for one hour.Next, add one microliter of CviQI enzyme to the reaction and incubate at 25 degrees Celsius for 30 minutes. When the RSAI and CviQI digestion is complete, proceed immediately with blunt ending. Prepare a 4.5 microliter mixture containing 2.5 microliters of DNA polymerase I, Large Fragment, and 2 microliters of 10 millimolar dNTPs.Transfer 1 microliter of the mixture to the DNA sample. The total volume will be 102 microliters. Mix well by vortexing and incubate at 25 degrees Celsius for one hour.Immediately proceed with streptavidin bead binding when the incubation is complete. Prepare the streptavidin beads by briefly vortexing the bead solution and transferring 50 microliters to a new two milliliter microcentrifuge tube. Remove the supernatant using a magnetic stand and wash the beans with 200 microliters of binding solution.Resuspend the beads in 100 microliters of binding solution and place the tube away from the magnetic stand. Transfer 100 microliters of the sample DNA to the 100 microliters of resuspended bead solution and mix carefully by pipetting to avoid any foaming of the solution. Incubate the tube at room temperature for 3 hours on a rotating wheel.Use the magnetic stand to capture the beads and discard the supernatant. Wash the beads twice in 400 microliters of washing solution and twice in 400 microliters of 1X T4 DNA ligase buffer. Resuspend the beads in 200 microliters of 1X T4 DNA ligase buffer and place it away from the magnetic stand.Linker ligation should be performed immediately after the streptavidin bead binding. Prepare a 400 microliter ligation reaction solution by mixing the following in a two milliliter microcentrifuge tube. 0.5 microliters of the DNA linker, 10 microliters of 10X T4 DNA ligase buffer, 20 microliters of 5X T4 DNA ligase buffer, 5 microliters of T4 DNA ligase, 164.5 microliters of nuclease free water, and 200 microliters of the resuspended beads.Place the reaction tube on a rotating wheel at room temperature for 3 hours. Wash the beads twice with the washing solution, and twice with 1X thermostable DNA polymerase buffer. Resuspend the beads in 50 microliters of 1X thermostable DNA polymerase PCR buffer, and place it away from the magnetic stand.To begin this procedure prepare a 200 microliter PCR reaction by adding the following to the resuspended beads. 10 microliters of one 1L-primer, 10 microliters of 1R-primer, 20 microliters of primer Link1, 10 microliters of 10X thermostable DNA polymerase buffer, four microliters of 10 milimolar dNTPs, eight microliters of thermostable DNA polymerase, and 88 microliters of nuclease-free water. Aliquot the reaction mixture into four PCR tubes and carry out PCR with the following condition.94 degrees Celsius for 2 minutes, 25 cycles of 94 degrees Celsius for 20 seconds, 56 degrees Celsius for 25 seconds, and 72 degrees Celsius for two minutes, and a final extension at 72 degrees Celsius for 5 minutes. Pool all four PCR reactions into a 2 ml microcentrifuge tube. Follow the procedure of the PCR purification kit and elute with 50 microliters of elution buffer.Determine the DNA concentration and the ratio of absorbance at 260 and 280 nanometers using a UV visible spectrophotometer. The DNA can now be stored at minus 20 degrees Celsius until ready for the next step. Since the procedures for the left junction and right junction specific amplifications are identical except for the primers used in the PCR, only the left junction amplification will be demonstrated.Prepare a 50 microliter PCR reaction by mixing the following in a PCR tube. Five microliters of DNA, five microliters of 10 micromolar 2L-primer, five microliters of 10 micromolar primer Link2, 5 microliters of 10X thermostable DNA polymerase buffer, one microliter of 10 milimolar dNTPs, two microliters of thermostable DNA polymerase, and 27 microliters of nuclease-free water. Carry out PCR with the following condition.94 degrees Celsius for 3 minutes, 8 to 15 cycles of 94 degrees Celsius for 20 seconds, 56 degrees Celsius for 25 seconds, and 72 degrees Celsius for two minutes, and a final extension at 72 degrees Celsius for 5 minutes. Use the PCR purification kit to purify the DNA and elute with 50 microliters of elution buffer. Determine the DNA concentration and ratio of absorbance at 260 and 280 nanometers.The last step in generating left and right junction sequence libraries is to analyze the PCR Amplicon length variations by performing capillary electrophoresis. This representative result shows varying lengths of PCR Amplicons in lentiviral vector integrations sites for both the upstream and downstream vector host junctions. The DNA size marker is in the M lane.After sequencing the PCR Amplicons, computational integration site sequence analysis is performed using an in-house programming script. The bi-directional integration site assay generates different sizes of PCR Amplicons for both the upstream and downstream vector host junctions visualized by capillary electrophoresis. A comparison of percent integration in genes Alu and L1 repeats of lentiviral vectors in humanized mouse repopulating cells within silico generated random integration events revealed that integration sites are significantly enriched in genes.The relative detection frequencies of vector integromes were calculated using only the sequence counts of integration site junctions generating smaller than 500bp PCR Amplicons. Approximately 77%of the vectors could be quantitatively analyzed by the bi-directional approach. In the strategy for clonal quantification we presume that each individual clone shares the same vector integrome.The relative frequencies of the left and right junctions are combined to represent the relative quantities of the clonal populations, or QVI. This color scheme shows the relative frequencies of 44 QVI clones in humanized mouse repopulating cells. Based on in silico 10, 000 random integration analysis, a 1.25 fold overestimation of clonal frequencies is expected when using GTAC motif enzymes, and a 2.56 overestimation is expected when using a TCGA motif enzyme.Once mastered, this technique can be divided in parts and completed in two days, if performed properly. While attempting this procedure it is important to remember that PCR steps are extremely sensitive, and precautions should be take to avoid any contamination. This technique has paved the way to examining the safety of therapeutic retroviral vectors, and to explore long-term repopulation patterns of hemotopoietic stem cell clones following transplant in rhesus macaques.

bidirectional-retroviral-integration-site-pcr-methodology

Research

JoVE Journal

Genetics

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

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Sammanslagning Absolut och relativ kvantitativ PCR data för att kvantifiera STAT3 Splice Variant Avskrifter

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

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

Detektion av mikroRNA-uttryck i peritonealt membran hos råttor med användning av kvantitativ realtids-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

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Retroviral Scanning: Kartläggning av MLV-integrationswebbplatser för att definiera cellspecifika reglerande regioner

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

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

Provberedning och analys av RNASeq-baserade gen uttryck Data från 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

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

En standardmetod att undersöka Hotellets mutagenicitet som en funktion av punktmutation reparation katalyseras av CRISPR/Cas9 och SsODN i mänskliga 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

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.

Metodik för korrekt upptäckt av mitokondrie-DNA metylering

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

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

Datoriserad analys av sekvensdata från Batch jäst 2-Hybrid skärmar

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

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are then&#160;used to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

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

The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

Multi-Locus variabel-antal tandem-upprepa analys av den fisk-patogena bakterien yersinia ruckeri av MULTIPLEX PCR och kapillärelektrofores

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

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 perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

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

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

Digitalt PCR-baserat konkurrens index för högt dataflöde analys av lämplighet för salmonella

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

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

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

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

Kvantitativ RT-PCR med hög genomströmning i en- och bulk C. elegansprover med nanofluidisk teknik

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