Name: simple bulk readout of digital nucleic acid quantification assays
Uploaded: 2015-09-24T15:00:00
Description: Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

The authors acknowledge Liyi Xu for providing MDA reagents and protocols. We also thank David Feldman and Navpreet Ranu for discussions relating to this work. This work was supported in part by a Burroughs Wellcome Career Award at the Scientific Interface to PCB.

Bemærk: Bearbejdning af mikrofluidanordning er ikke nødvendigt for denne analyse, som dråber til denne protokol kan dannes med eksisterende kommercielle dråbe beslutningstagere 23,29. 1. Gør Droplet-dannende mikrovæskeanordning <ol><li> Forbered mastermodel for kanalerne med SU-8 Master Fabrication protokollen beskrevet tidligere 31, men med en dråbe generator maskemønster 32. </li><li> Fabrikere enheder i PDMS ved hjælp af teknikker til bl?...

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	<li>Sykes, P. J., Neoh, S. H., Brisco, M. J., Hughes, E., Condon, J., Morley, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+targets+for+PCR+by+use+of+limiting+dilution.">Quantitation of targets for PCR by use of limiting dilution.</a> Biotechniques. 13 (3), 444-449 (1992).</li><li>Kalinina, O., Lebedeva, I., Brown, J., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoliter+scale+PCR+with+TaqMan+detection.">Nanoliter scale PCR with TaqMan detection.</a> Nucleic Acids Res. 25 (10), 1999-2004 (1997).</li><li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proc. Natl. Acad. Sci. U.S.A.. 96 (16), 9236-9241 (1999).</li><li>Day, E., Dear, P. H., McCaughan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+strategies+in+the+development+and+analysis+of+molecular+biomarkers+for+personalized+medicine.">Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine.</a> Methods. 59 (1), 101-107 (2013).</li><li>Tatusova, T., Ciufo, S., Fedorov, B., O'Neill, K., Tolstoy, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RefSeq+microbial+genomes+database:+new+representation+and+annotation+strategy.">RefSeq microbial genomes database: new representation and annotation strategy.</a> Nucleic Acids Res. 42 (Database issue), D553-D559 (2014).</li><li>Blainey, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+is+now:+single-cell+genomics+of+bacteria+and+archaea.">The future is now: single-cell genomics of bacteria and archaea.</a> FEMS Microbiol. Rev. 37 (3), 407-427 (2013).</li><li>Blainey, P. C., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+MDA+for+enumeration+of+total+nucleic+acid+contamination.">Digital MDA for enumeration of total nucleic acid contamination.</a> Nucleic Acids Res. 39 (4), e19 (2011).</li><li>Blainey, P. C., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+genomic+diversity,+one+cell+at+a+time.">Dissecting genomic diversity, one cell at a time.</a> Nat. Methods. 11 (1), 19-21 (2014).</li><li>Binga, E. K., Lasken, R. S., Neufeld, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Something+from+(almost)+nothing:+the+impact+of+multiple+displacement+amplification+on+microbial+ecology.">Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.</a> ISME J. 2 (3), 233-241 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+the+Year+2013.">Method of the Year 2013.</a> Nature Methods. 11 (1), 1 (2014).</li><li>Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+digital+PCR+enables+multigene+analysis+of+individual+environmental+bacteria.">Microfluidic digital PCR enables multigene analysis of individual environmental bacteria.</a> Science. 314 (5804), 1464-1467 (2006).</li><li>Shen, F., Du, W., Kreutz, J. E., Fok, A., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+on+a+SlipChip..">Digital PCR on a SlipChip..</a> Lab Chip. 10 (20), 2666-2672 (2010).</li><li>Heyries, K. 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=Megapixel+digital+PCR.">Megapixel digital PCR.</a> Nat. Methods. 8 (8), 649-651 (2011).</li><li>Dressman, D., Yan, H., Traverso, G., Kinzler, K. W., Vogelstein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+single+DNA+molecules+into+fluorescent+magnetic+particles+for+detection+and+enumeration+of+genetic+variations.">Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (15), 8817-8822 (2003).</li><li>Kiss, M. 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=High-throughput+quantitative+polymerase+chain+reaction+in+picoliter+droplets.">High-throughput quantitative polymerase chain reaction in picoliter droplets.</a> Anal. Chem. 80 (23), 8975-8981 (2008).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+hits+its+stride.">Digital PCR hits its stride.</a> Nat. Methods. 9 (6), 3 (2012).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+picoliter+rt-PCR+assays+using+microfluidics.">Parallel picoliter rt-PCR assays using microfluidics.</a> Anal. Chem. 78 (3), 956-958 (2006).</li><li>Lee, 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=Synchronized+reinjection+and+coalescence+of+droplets+in+microfluidics.">Synchronized reinjection and coalescence of droplets in microfluidics.</a> Lab Chip. 14 (3), 509-513 (2014).</li><li>Rhee, 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=Pressure+stabilizer+for+reproducible+picoinjection+in+droplet+microfluidic+systems.">Pressure stabilizer for reproducible picoinjection in droplet microfluidic systems.</a> Lab Chip. 14 (23), 4533-4539 (2014).</li><li>Hatch, A. 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=1-Million+droplet+array+with+wide-field+fluorescence+imaging+for+digital+PCR.">1-Million droplet array with wide-field fluorescence imaging for digital PCR.</a> Lab Chip. 11 (22), 3838-3845 (2011).</li><li>Hindson, B. 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=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Anal. Chem. 83 (22), 8604-8610 (2011).</li><li>Guo, M. T., Rotem, A., Heyman, J. A., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+microfluidics+for+high-throughput+biological+assays.">Droplet microfluidics for high-throughput biological assays.</a> Lab Chip. 12 (12), 2146-2155 (2012).</li><li>Lage, J. 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=Whole+genome+analysis+of+genetic+alterations+in+small+DNA+samples+using+hyperbranched+strand+displacement+amplification+and+array-CGH.">Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.</a> Genome Res. 13 (2), 294-307 (2003).</li><li>Dean, F. 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=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (8), 5261-5266 (2002).</li><li>Zong, C., Lu, S., Chapman, A. R., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+detection+of+single-nucleotide+and+copy-number+variations+of+a+single+human+cell.">Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.</a> Science. 338 (6114), 1622-1626 (2012).</li><li>Abate, A. R., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syringe-vacuum+microfluidics:+A+portable+technique+to+create+monodisperse+emulsions.">Syringe-vacuum microfluidics: A portable technique to create monodisperse emulsions.</a> Biomicrofluidics. 5, 14107 (2011).</li><li>Eyer, K., Kuhn, P., Stratz, S., Dittrich, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+chip+for+the+versatile+chemical+analysis+of+single+cells.">A microfluidic chip for the versatile chemical analysis of single cells.</a> J Vis Exp. (80), e50618 (2013).</li><li>Fainman, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optofluidics : fundamentals, devices, and applications. , (2010).</li><li>Xia, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G.M.+Softlithographie.">G.M. Softlithographie.</a> Angew Chem. 110 (5), 26 (1998).</li><li>Woyke, 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=Decontamination+of+MDA+reagents+for+single+cell+whole+genome+amplification.">Decontamination of MDA reagents for single cell whole genome amplification.</a> PLoS One. 6 (10), e26161 (2011).</li><li>Hindson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System+for+hot-start+amplification+via+a+multiple+emulsion.">System for hot-start amplification via a multiple emulsion.</a> US patent. , (2014).</li><li>Utada, A. 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=Monodisperse+double+emulsions+generated+from+a+microcapillary+device.">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308 (5721), 537-541 (2005).</li><li>Eastburn, D. J., Sciambi, A., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picoinjection+enables+digital+detection+of+RNA+with+droplet+rt-PCR.">Picoinjection enables digital detection of RNA with droplet rt-PCR.</a> PLoS One. 8 (4), e62961 (2013).</li><li>Dube, S., Qin, J., Ramakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+analysis+of+copy+number+variation+in+a+DNA+sample+using+digital+PCR+on+a+nanofluidic+device.">Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device.</a> PLoS One. 3 (8), e2876 (2008).</li><li>Shen, 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=Multiplexed+quantification+of+nucleic+acids+with+large+dynamic+range+using+multivolume+digital+RT-PCR+on+a+rotational+SlipChip+tested+with+HIV+and+hepatitis+C+viral+load.">Multiplexed quantification of nucleic acids with large dynamic range using multivolume digital RT-PCR on a rotational SlipChip tested with HIV and hepatitis C viral load.</a> J Am. Chem. Soc. 133 (44), 17705-17712 (2011).</li><li>Link, D. R., Anna, S. L., Weitz, D. A., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometrically+mediated+breakup+of+drops+in+microfluidic+devices.">Geometrically mediated breakup of drops in microfluidic devices.</a> Phys. Rev. Lett. 92 (5), 054503 (2004).</li><li>Nisisako, T., Torii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration+on+a+chip+for+mass+production+of+monodisperse+droplets+and+particles.">Microfluidic large-scale integration on a chip for mass production of monodisperse droplets and particles.</a> Lab Chip. 8 (2), 287-293 (2008).</li><li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <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+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device.">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802-807 (2012).</li></ol>

Digitale assays er kraftfulde metoder, der muliggør detektion af sjældne celler og tælling af de enkelte af nukleinsyremolekyler. Imidlertid digitale assays stadig ikke rutinemæssigt anvendes i analyselaboratorier, dels på grund af udgifterne til specialudstyr forbundet med kommercielt tilgængelige metoder. Her beskriver vi et endepunkt digital assay til kvantificering af nukleinsyrer med en forenklet analog udlæsning ved anvendelse af en standard realtid kvantitativ PCR maskine. Denne metode er hurtig at sætte ...

Digitale assays for nukleinsyre kvantificering (digital PCR) 1-4 og base rækkefølge (sekventering) er stærkt påvirker biovidenskab og medicin. Digitale analyser giver kvantificering af molekylære tællinger på en absolut skala (ikke i forhold til en kontrol), der giver høj følsomhed, så overfladiske sammenligninger på tværs af eksperimenter, og afgørende, så opførelsen af store databaser, der indeholder sammenlignelige data 5 (tabel 1). Over de sidste 15 år, hel genomamplifikation (WGA) opstået sammen med PCR som et generelt værktøj til nukleinsyreamplifikation. Ligesom PCR, WGA er nyttigt for analytiske og præparative applikationer ved at forstærke minut prøve elementer op til et niveau, der nemt kan opdages eller anvendes til efterfølgende analyser som basis sekventering. I modsætning til PCR, WGA er ikke specifikke for en bestemt DNA-locus, snarere tillader opformering af alle sekvenser i prøven, herunder ukendte sekvenser.Denne grundlæggende forskel mellem PCR og WGA gør de metoder, der supplerer hinanden og giver anledning til forskellige udfordringer i deres ansøgning. Den højt udbytte af WGA reaktioner 6 muliggør rutinemæssig opformering af genomisk DNA fra enkelte molekyler 7, enkeltceller 8 og andre lav biomasse prøver 9 til kvantificering eller yderligere analyse. De største udfordringer forbundet med WGA kemi er dens ekstreme følsomhed over for forureninger og den ujævne forstærkning tværs individuelle skabelon molekyler 6. Dog er WGA stigende popularitet som encellede sekventering har vist sig som den &quot;killer application&quot; af WGA-teknologi 10 og skabelon kvantificering af WGA er vigtig i mange anvendelsesområder 7. Instrumentering til digital nukleinsyre kvantificering er tidligere blevet beskrevet i en række ventil og ventilløs mikrofluid formats 11-14, herunder dråbe-baserede assays 15,16 (tabel 2). Men kommercielle mikrofluide systemer til digital analyse kræver specialudstyr til reaktion opsætning og produkt registrering 17. Brugerdefineret ventilforsynede mikrofluidik er fleksible, men kræver præcision mikrofabrikation og pneumatiske kontrolsystemer 18. Selv om det er relativt enkelt at gøre monodisperse mikro-dråber til emulsion-baserede digitale assays 19,20, digital udlæsning er teknisk besværlig, kræver enten store Vidvinkelbilledet imaging (svarende til populære næste generations sekventering teknologier) 21,22 eller højhastigheds-flow-baserede dråbe afsløring 23-25. Ideelt set ville en digital assay være enkel fra set-up til udlæsning, hvilket reducerer behovet for komplekse instrumentering og tillade et stort antal prøver, der skal udlæses hurtigt. Her beskriver vi en forenklet metode til udlæsning af digitale droplet assays, der bruger et konventionelt realtids-PCR instrument at måle fluorescens hovedparten af ​​dråber-baserede digitale assays. Selv om den nye metode kan anvendes til digitale PCR-assays, er det særligt fordelagtigt for digitale WGA assays for hvilke analog realtid amplifikationsassays (som giver gode resultater for PCR) er problematiske. WGA er almindeligt anvendt på prøver med template molekyler, der er heterogene i rækkefølge, længde og baseindhold. Disse forskellige templatemolekyler amplificeres ved forskellige hastigheder 6, hvilket nødvendiggør anvendelsen af en reference (&quot;standard&quot;) prøve med matchende egenskaber. Ofte sådan standard ikke er tilgængelig, eller egenskaberne ved de indkommende prøver er ukendte. Heterogenitet input materiale og længde-afhængig egenskab ved WGA kemier 26 også komplicere fortolkning af resultater ved at skabe uklarhed i, hvad der bliver kvantificeres - input masse, input antal molekyler, en kombination af de to, eller ingen af delene. Finally, sekvensen-ikke-specificitet gør kvantitativ forstærkning multipel forskydning (MDA) mere følsomme over for forurening end kvantitativ PCR siden forurenende molekyler af enhver sekvens har et potentiale til at blande sig. Mikrofluide digitale assays behandle forurening med adskillelse templatemolekyler og reducere reaktionsvolumener, således at færre kontaminanter samples. Her bruger vi en populær isotermiske WGA metode, MDA 27. Notatet flere andre WGA kemier herunder PicoPlex og MALBAC 28 afhænger afgørende af indledende skridt isoterm strengdeplacering. Isotermiske trin forværre den udfordring at anvende analoge realtid analyser til kvantitativ WGA. WGA kan hverken helt forhindres under opsætningen, hvilket fører til uønsket præ-variabel forstærkning, eller diskretiseres (&quot;cykluser&quot;) på en måde, replikation af heterogene molekyler kan drives til færdiggørelse og stoppet før den næste cyklus som PCR 11 </sup&gt; (figur 1). En digital assay format til WGA rumme typiske reaktion opsætning på grund af adskillelsen af ​​hvert molekyle til tælling ved analysen endpoint (så pre-forstærkning påvirker ikke resultaterne) original udlæsning betyder nøjagtighed ville være stort set uafhængig af variationer i forstærkning effektivitet. Analysen afhænger af dråber produceret i olie med ensartede mængder, som signalniveauet pr dråbe ved analysen endepunkt vil afhænge af dråben volumen, og vi ønsker ikke konsistensen af ​​resultaterne til at afhænge af gennemsnit på tværs af en fordeling af dråbestørrelser. Making monodisperse dråber er nu en standardprocedure (ca. 3.500 monodisperse dråbe papirer er blevet offentliggjort siden 2013), men kræver mikrofluid instrumentering 25. Faktisk har mere end seks selskaber udviklet selvstændige kommercielle produkter, der er afhængige af produktionen af ​​sådanne dråber, og dråbe-making mikrofluide chips erkommercielt tilgængelig 23,29. Til denne undersøgelse anvendte vi tilpassede mikrofluidenheder fremstillet in-house (se protokol). Sprøjtepumper at drive strømmen gennem enhederne er også kommercielt tilgængelige, men kan alternativt være substitueret med en enkelt engangssprøjte til vakuum-drevet flow at reducere omkostningerne 30.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Evagreen Dye</td>
			<td>Biotium</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>New England Biotechnologies</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phi29 DNA Polymerase Reaction Buffer</td>
			<td>New England Biotechnologies</td>
			<td>B0269S</td>
			<td>Vortex periodically until aggregate dissolves</td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>New England Biotechnologies</td>
			<td>N3011S</td>
			<td>Heated to 50C and quenched on ice prior to dilution</td>
		</tr>
		<tr>
			<td>Randomized MDA oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#39;-NNNNN*N-3&#39;</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#8217;-CGGCAAACGGGAATGAAACGCC-3&#8217; and 5&#8217;-TGCGGCAAAGACAGCAACGG-3&#8217;</td>
		</tr>
		<tr>
			<td>UltraPure Nuclease-free water</td>
			<td>Life Technologies</td>
			<td>10977-015</td>
			<td>UV-treated for 30m in Stratalinker 2400 before use (optional)</td>
		</tr>
		<tr>
			<td>ROX reference dye</td>
			<td>Life Technologies</td>
			<td>12223-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR optical strip caps</td>
			<td>Life Technologies</td>
			<td>4323032</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Agilent Technologies</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP (25uM each)</td>
			<td>Agilent Technologies</td>
			<td>200415</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler - Mx3000P qPCR system</td>
			<td>Agilent Technologies</td>
			<td>401403</td>
			<td></td>
		</tr>
		<tr>
			<td>Barrier pipette tips</td>
			<td>VWR</td>
			<td>89003-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-rad oil for evagreen</td>
			<td>Bio-rad</td>
			<td>186-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>JumpStart Taq ReadyMix</td>
			<td>Sigma-Aldrich</td>
			<td>P2893-100RXN</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple bulk readout of digital nucleic acid quantification assays

Digitale assays er kraftfulde metoder, der muliggør detektion af sjældne celler og tælling af individuelle nukleinsyremolekyler. Men digitale assays er stadig ikke rutinemæssigt anvendes, på grund af omkostningerne og særligt udstyr forbundet med kommercielt tilgængelige metoder. Her præsenteres en forenklet metode til udlæsning af digitale droplet assays under anvendelse af en konventionel realtids-PCR instrument til måling hovedparten fluorescens af dråber-baserede digitale assays. Vi karakteriserer ydeevnen af ​​bulk udlæsning assay under anvendelse af syntetiske dråbe blandinger og en dråbe digitalforstærkning multiple forskydning (MDA) assay. Kvantitativ MDA især fordele fra et digitalt reaktion format, men vores nye metode gælder for alle digitale assay. For etablerede digitale assayprotokoller såsom digital PCR, denne metode tjener til at fremskynde og forenkle analysen udlæsning. Vores hovedparten udlæsning metode bringer fordelene ved partitioned analyser uden behov for specialiseret udlæsning instrumentering. De vigtigste begrænsninger hovedparten udlæsning metode reduceres dynamikområde sammenlignet med dråbe-optælling platforme og behovet for en standardprøve, selv om kravene til denne standard er mindre krævende end for en konventionel realtid eksperiment. Kvantitativ hele genomamplifikation (WGA) anvendes til at teste for forurenende stoffer i WGA reaktioner og er den mest følsomme måde at detektere tilstedeværelsen af ​​DNA-fragmenter med ukendte sekvenser, der giver fremgangsmåden meget lovende i forskellige anvendelsesområder herunder farmaceutiske kvalitetskontrol og astrobiologi.

Mens konventionelle bulk / realtid udlæsninger kan anvendes til både kvantitativ PCR og kvantitative WGA assays (figur 1), digitale kvantitative assays tilvejebringer fordele (tabel 1). I den beskrevne metode, vi læser ud digitale analyser i mikro-dråbe-format med en simpel bulk-endpoint måling (figur 2). Selv om denne fremgangsmåde er bredt anvendelig har vi fokus på kvantitativ WGA (MDA), fordi denne metode giver særlige udfordringer for konventionelle realtid...

Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Enkel Bulk Udlæsning af digitale Nucleic Acid Kvantificering Analyser

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

simple-bulk-readout-of-digital-nucleic-acid-quantification-assays

Research

JoVE Journal

Biology

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

El-Free, Sekventiel Nucleic Acid og protein isolering

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

Analysere og Building nukleinsyrestrukturer med 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZ Polymerization Analyser: enkle protokoller og overvejelser

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

Forberedelse, Imaging, og kvantificering af bakteriel Surface Motilitet assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Effektiv Nucleic Acid Udvinding og 16S rRNA Gene sekventering for bakteriel Community karakterisering

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtrering Isolering af nukleinsyrer: en enkel og hurtig DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

Laktat Dehydrogenase Sequestration Assay — En simpel og pålidelig metode til at bestemme Bulk Autophagic beslaglæggelse aktivitet i pattedyrceller

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Planarian Scrunching som en kvantitativ behavioral udlæsning for skadelige Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

To-trins omvendt transkriptionsdråbe digitale PCR-protokoller til SARS-CoV-2-detektion og kvantificering

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...