Name: the visual colorimetric detection of multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform
Uploaded: 2016-09-27T14:00:00
Description: Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

Ministry of Science and Technology of Taiwan provided financial support of this research under contracts MOST-103-2221-E-002 -097 -MY3.

1. metod för att upptäcka MNP Obs: Detta avsnitt beskriver proceduren för att detektera MNP baserat på hybridisering-medierad tillväxt av guldnanopartiklar. <ol><li> Bereda sond-DNA (5&#39;-tiol-GAGCTGGTGGCGTAGGCAAG-3 &#39;) lösning vid en koncentration 100 pM. </li><li> Förbered prob-DNA-modifierad AuNP (AuNP sond) partiklar. 21 Obs: Volymen som används här beror på kravet på prob-DNA-modifierad AuNP (AuNP sond) partiklar. Det är därmed beroende av antalet experiment som skall u...

<ol>
	<li>Chorley, B. 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=Discovery+and+verification+of+functional+single+nucleotide+polymorphisms+in+regulatory+genomic+regions:+current+and+developing+technologies.">Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: current and developing technologies.</a> Mutat. Res. Rev. Mutat. 659 (1), 147-157 (2008).</li><li>Hinds, D. 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=Whole-genome+patterns+of+common+DNA+variation+in+three+human+populations.">Whole-genome patterns of common DNA variation in three human populations.</a> Science. 307 (5712), 1072-1079 (2005).</li><li>Rosenfeld, J. A., Malhotra, A. K., Lencz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+multi-nucleotide+polymorphisms+in+the+human+genome+characterized+by+whole+genome+and+exome+sequencing.">Novel multi-nucleotide polymorphisms in the human genome characterized by whole genome and exome sequencing.</a> Nucleic Acids Res. , 408 (2010).</li><li>Whelan, S., Goldman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+frequency+of+events+that+cause+multiple-nucleotide+changes.">Estimating the frequency of events that cause multiple-nucleotide changes.</a> Genetics. 167 (4), 2027-2043 (2004).</li><li>Saiki, R. K., Walsh, P. S., Levenson, C. H., Erlich, 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=Genetic+analysis+of+amplified+DNA+with+immobilized+sequence-specific+oligonucleotide+probes.">Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.</a> Proc. Natl. Acad. Sci. U.S.A. 86 (16), 6230-6234 (1989).</li><li>Kim, S., Misra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping:+technologies+and+biomedical+applications.">SNP genotyping: technologies and biomedical applications.</a> Annu. Rev. Biomed. Eng. 9, 289-320 (2007).</li><li>Kwok, P. Y., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+single+nucleotide+polymorphisms.">Detection of single nucleotide polymorphisms.</a> Curr. Issues Mol. Biol. 5, 43-60 (2003).</li><li>Elghanian, R., Storhoff, J. J., Mucic, R. C., Letsinger, R. L., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+colorimetric+detection+of+polynucleotides+based+on+the+distance-dependent+optical+properties+of+gold+nanoparticles.">Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles.</a> Science. 277 (5329), 1078-1081 (1997).</li><li>Lee, J. S., Han, M. S., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+Detection+of+Mercuric+Ion+(Hg2+)+in+Aqueous+Media+using+DNA-Functionalized+Gold+Nanoparticles.">Colorimetric Detection of Mercuric Ion (Hg2+) in Aqueous Media using DNA-Functionalized Gold Nanoparticles.</a> Angew. Chem. 119 (22), 4171-4174 (2007).</li><li>Storhoff, J. 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=Gold+nanoparticle-based+detection+of+genomic+DNA+targets+on+microarrays+using+a+novel+optical+detection+system.">Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system.</a> Biosens. Bioelectron. 19 (8), 875-883 (2004).</li><li>Bao, Y. P., 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=SNP+identification+in+unamplified+human+genomic+DNA+with+gold+nanoparticle+probes.">SNP identification in unamplified human genomic DNA with gold nanoparticle probes.</a> Nucleic Acids Res. 33 (2), 15-15 (2005).</li><li>Chen, Y. T., Hsu, C. L., Hou, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+single-nucleotide+polymorphisms+using+gold+nanoparticles+and+single-strand-specific+nucleases.">Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.</a> Anal. Biochem. 375 (2), 299-305 (2008).</li><li>Li, H., Rothberg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+of+DNA+sequences+based+on+electrostatic+interactions+with+unmodified+gold+nanoparticles.">Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (39), 14036-14039 (2004).</li><li>Fang, W. F., Chen, W. J., Yang, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+determination+of+DNA+concentration+and+mismatches+using+hybridization-mediated+growth+of+gold+nanoparticle+probes.">Colorimetric determination of DNA concentration and mismatches using hybridization-mediated growth of gold nanoparticle probes.</a> Sensor. Actuat. B Chem. 192, 77-82 (2014).</li><li>Wang, Z., Zhang, J., Ekman, J. M., Kenis, P. J., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-mediated+control+of+metal+nanoparticle+shape:+one-pot+synthesis+and+cellular+uptake+of+highly+stable+and+functional+gold+nanoflowers.">DNA-mediated control of metal nanoparticle shape: one-pot synthesis and cellular uptake of highly stable and functional gold nanoflowers.</a> Nano Lett. 10 (5), 1886-1891 (2010).</li><li>Chen, L., Manz, A., Day, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+nucleic+acid+analysis+integrated+on+microfluidic+devices.">Total nucleic acid analysis integrated on microfluidic devices.</a> Lab Chip. 7 (11), 1413-1423 (2007).</li><li>Lien, K. Y., Liu, C. J., Lin, Y. C., Kuo, P. L., Lee, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+genomic+DNA+and+detection+of+single+nucleotide+polymorphism+genotyping+utilizing+an+integrated+magnetic+bead-based+microfluidic+platform.">Extraction of genomic DNA and detection of single nucleotide polymorphism genotyping utilizing an integrated magnetic bead-based microfluidic platform.</a> Microfluid. Nanofluid. 6 (4), 539-555 (2009).</li><li>Ng, J. K. K., Feng, H. H., Liu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+discrimination+of+single-nucleotide+mismatches+using+a+microfluidic+device+with+monolayered+beads.">Rapid discrimination of single-nucleotide mismatches using a microfluidic device with monolayered beads.</a> Anal. Chim. Acta. 582 (2), 295-303 (2007).</li><li>Xing, S., Harake, R. S., Pan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-driven+transports+on+superhydrophobic-patterned+surface+microfluidics.">Droplet-driven transports on superhydrophobic-patterned surface microfluidics.</a> Lab Chip. 11 (21), 3642-3648 (2011).</li><li>Huang, C. J., Fang, W. F., Ke, M. S., Chou, H. Y. E., Yang, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biocompatible+open-surface+droplet+manipulation+platform+for+detection+of+multi-nucleotide+polymorphism.">A biocompatible open-surface droplet manipulation platform for detection of multi-nucleotide polymorphism.</a> Lab Chip. 14 (12), 2057-2062 (2014).</li><li>Mirkin, C. A., Letsinger, R. L., Mucic, R. C., Storhoff, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA-based+method+for+rationally+assembling+nanoparticles+into+macroscopic+materials.">A DNA-based method for rationally assembling nanoparticles into macroscopic materials.</a> Nature. 382 (6592), 607-609 (1996).</li><li>Ichimura, K., Oh, S. K., Nakagawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-driven+motion+of+liquids+on+a+photo-responsive+surface.">Light-driven motion of liquids on a photo-responsive surface.</a> Science. 288 (5471), 1624-1626 (2000).</li><li>Jones, T. B., Gunji, M., Washizu, M., Feldman, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoretic+liquid+actuation+and+nanodroplet+formation.">Dielectrophoretic liquid actuation and nanodroplet formation.</a> J. Appl. Phys. 89 (2), 1441-1448 (2001).</li><li>Lee, J., Kim, C. J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-tension-driven+microactuation+based+on+continuous+electrowetting.">Surface-tension-driven microactuation based on continuous electrowetting.</a> J. Microelectromech. S. 9 (2), 171-180 (2000).</li><li>Daniel, S., Chaudhury, M. K., De Gennes, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibration-actuated+drop+motion+on+surfaces+for+batch+microfluidic+processes.">Vibration-actuated drop motion on surfaces for batch microfluidic processes.</a> Langmuir. 21 (9), 4240-4248 (2005).</li><li>Darhuber, A. A., Valentino, J. P., Davis, J. M., Troian, S. M., Wagner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+actuation+by+modulation+of+surface+stresses.">Microfluidic actuation by modulation of surface stresses.</a> Appl. Phys. Lett. 82 (4), 657-659 (2003).</li></ol>

I detta protokoll, till en enkel kolorimetrisk metod upptäcker MNP kan genomföras vid koncentrationer som sträcker sig från 0.11-0.50 iM i mikrocentrifugrör. Dessutom är den föreslagna MNP detekteringsmetod genomförs på ett pneumatiskt dropp manipulation plattform som har en hög potential för DNA screening och andra biomedicinska tillämpningar. I praktiken den detekterbara intervallet för prov-DNA koncentrationen beror på blandningseffektiviteten av de operativplattformar. För att säkerställa att samman...

Enbaspolymorfi (SNP), som är en enda baspars skillnad i en DNA-sekvens, är en av de vanligaste genetiska variationer. Aktuella studier rapporterar att SNP är förknippade med risk för sjukdom, drog effekt och biverkningar av individer genom att påverka geners funktion. 1,2 Nyligen genomförda studier visade också att två eller flera punktmutationer (multi-nucleotide polymorphism) orsaka vissa sjukdomar och individuell skillnader i effekterna av sjukdomen. 3,4 Upptäckten av nucleotide polymorphism är därför nödvändigt i prescreening sjukdom. Enkla och effektiva metoder för snabb detektion av sekvensspecifika oligonukleotider högt utvecklade under de senaste två decennierna. 1,5 Nuvarande metoder för att identifiera DNA-mutationer typiskt involverar förfaranden inklusive sond immobilisering, fluorescensmärkning, gelelektrofores, etc., 6,7 men dessa metoder kräver i allmänhet en lång analytiska processen, Expentande utrustning, välutbildade tekniker och betydande konsumtion av prover och reagenser. En nanopartikel med ett stort förhållande av ytarea till volym och unika fysikalisk-kemiska egenskaper är ett idealiskt material som en mycket känslig och billig detektering plattform för specifika biomarkörer. Guldnanopartiklar (AuNP) används ofta för DNA-detektion på grund av deras stora förmåga att ändras med oligonukleotidsonder. 8-10 SNP detekteringsteknik har också utvecklats med hjälp av AuNP. 11-13 I detta arbete har vi antagit en ny kolorimetrisk metod för att detektera multi-nucleotide polymorphism (MNP) genom DNA-hybridisering-förmedlad tillväxt av AuNP prober. 14 Denna enkla och snabba sondering metod är baserad på teorin att olika längder av enkelsträngat DNA (ssDNA) eller dubbelsträngat DNA (dsDNA) konjugerat till AuNP påverka tillväxten storleken och formen av den AuNP (se Figur 1). 15 Denna metod för DNA-detektionhar en liten förbrukning av reagens, en liten analys varaktighet (några minuter), och en enkel procedur utan värmereglering som är framåtriktat gäller för klinisk diagnos och inhemska hälsoundersökas. Flera mikroflödessystem för att upptäcka DNA-sekvensen har utvecklats, 16 de mikroflödessystem, utvecklats från traditionella experimentella protokoll, krävs färre bitar av storskalig utrustning och förenklat experimentella protokoll för att förbättra känsligheten, detektionsgräns och specificitet av DNA biosensor. Metoder DNA upptäckt i mikroflödessystem fortfarande kräver dock instrument en efterföljande process såsom PCR (polymerase chain reaction) maskin för signalförstärkning och en fluorescensläsare för en enda avläsning för att identifiera den heterogena SNP. 17,18 utveckla en enkel plattform utan efterföljande behandling för att direkt läsa ut resultaten av multi-nucleotide polymorphismär mycket önskvärd. Jämfört med väl använt, konventionell, slutna mikroflödessystem, den öppna ytan mikrofluidikanordningar lovande erbjuder flera fördelar, såsom en tydlig optisk väg, ett enkelt sätt att komma åt exemplet, en direkt miljö tillgänglighet och ingen lätt bildas kavitation eller gräns obstruktion kanalen. 19 Vårt tidigare arbete infört en enkel pneumatisk plattform för öppen yta dropp manipulation (se figur 2). 20 på denna plattform, droppar kan samtidigt transporteras och manipuleras utan inblandning från en drivande energi med hjälp av en sugkraft, som har en stor potential i biologiska och kemiska tillämpningar. Denna pneumatisk plattform var således utnyttjas för att exekvera manipulering av DNA-prover för MNP detektering i kombination med den kolorimetriska metod med hjälp av begreppet DNA-hybridisering-förmedlad tillväxt av AuNP prober. Protokollet presenteras i detta dokument beskriveren enkel visuell detektion av flera nucleotide polymorphisms på den pneumatiska dropp manipulation plattform på en öppen yta. Detta arbete bekräftar att fler nukleotidpolymorfism är detekterbar med blotta ögat; den föreslagna pneumatiska plattform är lämpliga för biologiska och kemiska tillämpningar.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">PDMS</td>
			<td style="width:204px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">benchtop engravers</td>
			<td style="width:204px;">&#160;Roland DG</td>
			<td style="width:119px;">EGX-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">laser cutting machine</td>
			<td style="width:204px;">Universal Laser Systems, Inc.</td>
			<td style="width:119px;">VLS 3.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Oxygen plasma treatment system</td>
			<td style="width:204px;">Femto Science Inc. Korea</td>
			<td style="width:119px;">CUTE-MPR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">solenoid valve</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td>home built</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">vacuum pump</td>
			<td>ULVAC KIKO, Inc.</td>
			<td style="width:119px;">DA-30D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">13-nm AuNP solution</td>
			<td style="width:204px;">TAN Bead Inc., Taiwan</td>
			<td style="width:119px;">NG-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNA (with 5 -end labeled thiol)</td>
			<td style="width:204px;">MDBio, Inc., Taiwan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">phosphate buffered saline (PBS)</td>
			<td>UniRegion&#160;Bio-Tech,. Taiwan</td>
			<td>PBS001-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium dodecyl sulfate (SDS)</td>
			<td style="width:204px;">J. T. baker</td>
			<td style="width:119px;">4095-04</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Hydroxylamine solution (NH2OH)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:119px;">467804</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Chloroauric acid (HAuCl4)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>G4022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium chloride (NaCl)</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">vortex mixer</td>
			<td style="width:204px;">Digisystem Laboratory Instruments Inc.</td>
			<td style="width:119px;">VM-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">centrifuge</td>
			<td style="width:204px;">Hermle Labortechnik GmbH.</td>
			<td style="width:119px;">Z 216 MK</td>
			<td></td>
		</tr>
	</tbody>
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the visual colorimetric detection of multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform

En enkel och visuell metod för att detektera fler nucleotide polymorphism (MNP) utfördes på en pneumatisk dropp manipulation plattform på en öppen yta. Detta sätt att kolorimetriska DNA detektion baserades på hybridisering-förmedlad tillväxt av guldnanopartiklar sonder (AuNP Probes). Tillväxt storlek och konfiguration av AuNP domineras av antalet DNA-prover hybridiserade med sondema. Baserat på de specifika storlek- och formberoende optiska egenskaperna hos nanopartiklar, är antalet felpassningar i ett prov-DNA-fragment till sonderna kunna diskrimineras. Testerna genomfördes via droppar som innehåller reagens och DNA-prover respektive, och transporterades och blandas på den pneumatiska plattform med kontrollerad pneumatisk sugning av den flexibla PDMS-baserade superhydrofoba membran. Droppar kan levereras samtidigt och just på en öppen yta på den föreslagna pneumatiska plattform som är mycket biokompatibla med ingen sida effect av DNA-prov inuti dropparna. Genom att kombinera de två föreslagna metoderna kan multi nucleotide polymorphism detekteras på sikt på den pneumatiska dropp manipulation plattform; ingen ytterligare instrument krävs. Proceduren från att installera de små dropparna på plattformen för att det slutliga resultatet tar mindre än fem minuter, mycket mindre än med befintliga metoder. Dessutom kräver denna kombinerade MNP detekterings tillvägagångssätt en provvolym av endast 10 ^ il i varje operation, som är anmärkningsvärt mindre än den hos en makro-system.

I detta arbete har tre DNA-prover testades med användning av en enkel och ny metod för detektering genom DNA-hybridisering-förmedlad tillväxt av AuNP prober. Sekvenserna för prob-DNA och DNA-prover av tre slag, specifikt cDNA (helt komplementär för att sondera DNA), TMDNA (tre baspar felparad DNA), och SixMDNA (sex baspar felparad DNA) anges i protokoll steg 1. de felpassningar till sonden av DNA-proven som testades här är båda i mitten segment av DNA-proven. Figur 6</s...

Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

Detta arbete presenterar en enkel och visuell metod för att upptäcka flera nucleotide polymorphisms på en pneumatisk dropp manipulation plattform. Med den föreslagna metoden, hela experimentet, inklusive dropp hantering och spårning av fler nucleotide polymorphisms, kan utföras i närheten av 23 ° C utan hjälp av avancerade instrument.

Visual Kolorimetrisk detektion av Multi-nucleotide polymorphisms på en pneumatisk Dropp Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

The overall goal of this procedure is to combine a simple method for visually detecting multi-nucleotide polymorphisms with a pneumatic droplet manipulation platform. The DNA detection platform is on an open surface and is straight forward to use for biochemical researchers. We investigated a simple tool to repeatedly detect genetic mutations and genetic diseases.It has greater potential helping us understand how genetic variation leads to disease. We combined two novel and simple methods. The multi-nucleotide polymorphism can be detected on the pneumatic droplet manipulation platform with the naked eye and without any additional instrument.The method for detecting multi-nucleotide polymorphism will be demonstrated by Tzu-Ming Wang, the post doctor in our group. To begin this procedure, prepare a 100 micromolar solution of probe DNA as indicated in the text protocol. Then, add the probe DNA to a one OD per milliliter solution of gold nanoparticles.Bring the final concentration of SDS to 0.01%and of PBS to 0.01 molar. Allow the solution to incubate for 20 minutes. Next, increase the concentration of sodium chloride to 0.05 molar by adding a solution of two molar sodium chloride and 0.01 molar PBS.Maintain the SDS concentration at 0.01%and let the solution incubate for anoher 20 minutes. Increase the concentration of sodium chloride in increments of 0.1 molar over 20 minute intervals until a final concentration of one molar is reached. Incubate the solution at 23 degrees Celsius overnight on a rotator.After the DNA samples and gold nanoparticles have become conjugated, centrifuge the solution at 7, 000 times g for 30 seconds. Remove the supernatant. Next, prepare a 25 nanomolar gold nanoparticle probe solution by resuspending the pellet in deionized water.Prepare target DNA samples as outlined in the text protocol. Add six microliters of gold nanoparticle probe solution into each DNA sample with sodium chloride. Using a vortex mixer, shake each sample solution for five minutes at 23 degrees Celsius.Then, add six microliters of 400 millimolar hydroxyl amine and six microliters of 25.4 millimolar chloroauric acid to each DNA sample solution. Set these samples aside as a steady change in coloration will occur over the next hour. the size, shape-dependent optical properties of the nanoparticle, the number of the mismatch in a sample DNA fragment typical can be discerned by eye.To begin fabricating the droplet manipulation platform, utilize a computer numerical controlled machine equipped with a 0.5 millimeter drill bit to produce PMMA-based mastermolds with microstructures. For the drill, use a feed speed of seven millimeters per second and a rotation rate of 26, 000 rpm, and inspect the molds after drilling. Use an air blower and deionized water to clean the surface of the mastermolds and remove any PMMA scrap.Following this, prepare a 10 to one mixture of PDMS base and curing agent. Degas the mixture in a desiccator until all air bubbles are removed. Then, pour the PDMS mixture into the mastermold.Bake for three hours at 60 degrees Celsius to obtain the inverse microstructures in the air chambers and air channels. After baking is complete, remove the PDMS layer from the mastermold, and punch holes in it for the air suction inlets. Use an oxygen plasma treatment system to clean the PDMS membrane and PDMS layer.After treatment, bond the PDMS membrane and the PDMS layer, and then punch holes in this conjugated structure. Next, bond the PDMS and glass substrate together using a hot plate at 90 degrees Celsius for 10 minutes. Once the layers are bonded together, place the composite plate into a laser cutting machine PDMS membrane-side up.Import the dwg file to define the super hydrophobic area, and note this will be directly engraved into the PDMS membrane. Clean the newly engraved superhydrophobic surface with deionized water. This will wash away any carbonized residues.After the surface has been cleaned, connect the air suction inlet and vacuum pump through a solenoid valve. Then, connect the solenoid valve to a computer to control it with a digital IO USB module. Once the pneumatic droplet manipulation platform has been set up, add 10 microliters of 0.5 micromolar target DNA on the superhydrophobic area.Next, add 10 microliters of freshly hybridized 25 nanomolar gold nanoparticle probe solution next to the target DNA drop. Use a simple program to control air suction to the platform through a solenoid valve. Set the pressure to negative 80 kilopascals, and the driving frequency to five hertz.Provide suction along the droplet's vibration paths, causing them to collide and coalesce. Next, mix the coalesced droplet by manipulating the suction to roll the sample for three minutes. Add two microliters of 400 milimolar hydroxylamine, and two microliters of 25.4 milimolar chloroauric acid to the superhydrophobic area of the pneumatic droplet manipulation platform.Manipulate the suction so that the droplets collide and merge. Then, mix the merged droplet by rolling it for one minute. Observe the color of the final droplet with the naked eye.In this study, multi nucleotide polymorphisms are detected colorometrically using DNA hybridization mediated growth of gold nanoparticle probes. Here, the DNA samples possess different affinities to the probe DNA, causing differences in the growth of the gold nanoparticles, seen as a change in color. The three base pair mismatched DNA sample has less hybridization than the fully complimentary DNA, which can be seen as noticeable distinctions in color, especially at higher concentrations.The six base mismatched DNA has weak hybridization affinity to the probe, and thus the growth size of the gold nanoparticles was small. This colorometric analysis is shown to be repeatable when samples are manipulated via a pneumatic suction platform, as the results of DNA mismatch are readily observable by the naked eye. Once mastered, the entire procedure takes less than five minutes from the addition of the sample and the reagent until result of them, which is much quicker than the traditional method.The total in each operation is only 10 microliter, which is significantly less than what is required in a large system. Following this procedure, once the sequence variation of the space of DNA fragment is confirmed to be responsible for a specific disease. The specifically designed product can be used to acquire a number of mismatch of the sample DNA.The pneumatic droplet manipulation platform is more biocompatible than other methods such as optical weighting, dye electro fluorescence, electro weighting, and shoomoo's capillary actuation. The proposed multinucleotide polymorphism detection method was conducted under pneumatical droplet manipulation platform, which means the samples and reagents can be directly loaded and collected with pipette. After its development, this technique has a high potential for DNA screening, and other chemical and biomatical applications.

Research

JoVE Journal

Genetics

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