Name: primer-free aptamer selection using a random dna library
Uploaded: 2010-07-26T17:00:00
Duration: 11 min 14 s
Description: Watch this Scientific Journal Video about Primer-Free Aptamer Selection Using A Random DNA Library at JoVE.com

Wij danken Craig Paul op de Core Microarray faciliteit voor zijn hulp. Dit werk werd ondersteund door NIH / NCI subsidie ​​# CA118591 van de IMAT Program. SLD erkent ook een Pennsylvania Space Grant Consortium Fellowship voor financiële steun. Deze publicatie werd ook gesteund door de Pennsylvania State University Materials Research Institute Nano Fabrication Network en de National Science Foundation Cooperative arbeidsovereenkomst nr. 0335765, National Nanotechnology Infrastructure Network, met Cornell University.

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S., Sullenger, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+RNA+aptamers.">Gene therapy progress and prospects: RNA aptamers.</a> Gene Ther. 14, 283-2891 (2007).</li><li>Cowperthwaite, M. C., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatic+analysis+of+the+contribution+of+primer+sequences+to+aptamer+structures.">Bioinformatic analysis of the contribution of primer sequences to aptamer structures.</a> J Mol Evol. 67, 95-102 (2008).</li><li>Shtatland, T., Gill, S. C., Javornik, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Escherichia+coli+RNA+with+bacteriophage+MS2+coat+protein:+genomic+SELEX.">Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX.</a> Nucleic Acids Res. 28, E93-E93 (2000).</li><li>Jarosch, F., Buchner, K., Klussmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+using+a+dual+RNA+library+that+allows+primerless+selection.">In vitro selection using a dual RNA library that allows primerless selection.</a> Nucleic Acids Res. 34, E86-E86 (2006).</li><li>Vater, A., Jarosch, F., Buchner, K., Klussmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bioactive+Spiegelmers+to+migraine-associated+calcitonin+gene-related+peptide+rapidly+identified+by+a+novel+approach:+tailored-SELEX.">Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX.</a> Nucleic Acids Res. 31, E130-E130 (2003).</li><li>Wen, J. D., Gray, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+genomic+sequences+that+bind+tightly+to+Ff+gene+5+protein:+primer-free+genomic+SELEX.">Selection of genomic sequences that bind tightly to Ff gene 5 protein: primer-free genomic SELEX.</a> Nucleic Acids Res. 32, E182-E182 (2004).</li><li>Musheev, M. U., Krylov, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+aptamers+by+systematic+evolution+of+ligands+by+exponential+enrichment:+addressing+the+polymerase+chain+reaction+issue.">Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue.</a> Anal Chim Acta. 564, 91-916 (2006).</li><li>Pan, W. H., Clawson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer-free+aptamer+selection+using+a+random+DNA+library.">Primer-free aptamer selection using a random DNA library.</a> Methods Mol Biol. , (2009).</li><li>Pan, W., Xin, P., Clawson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal-Primer+and+Primer-Free+SELEX+Protocols+for+Selection+of+Aptamers+from+Random+DNA+Libraries.">Minimal-Primer and Primer-Free SELEX Protocols for Selection of Aptamers from Random DNA Libraries.</a> BioTechniques. 44, 351-360 (2008).</li><li>Nicewarner-Pena, S., Freeman, G., Reiss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicrometer+metallic+barcodes.">Submicrometer metallic barcodes.</a> Science. 294, 137-141 (2001).</li><li>JC, H. u. l. t. e. e. n., CR, M. a. r. t. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+template-based+method+for+the+preparation+of+nanomaterials.">A general template-based method for the preparation of nanomaterials.</a> J Materials Chem. 7, 1075-1087 (1997).</li></ol>

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<td>HotMaster TAQ DNA Polymerase</td>
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<td>5-Prime</td>
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<td>dNTP Mix</td>
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<td>Sigma</td>
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<td>Acrylamide</td>
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<td>Fisher</td>
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<td>UltraPure 10X TBE Buffer</td>
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<td>Invitrogen</td>
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<td>Ammonium Persulfate</td>
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<td>Bio-Rad</td>
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<td>TEMED</td>
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<td>Fisher</td>
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<td>PhiX174 DNA/Hinf I DNA Marker</td>
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<td>Promega</td>
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<td>Ethidium Bromide</td>
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<td>&alpha;-32P-dCTP</td>
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<td>Phenol:ChCl3:IAA</td>
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<td>Ambion</td>
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<td>NaCl</td>
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<td>Ethanol</td>
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<td>Restriction Enzymes</td>
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<td>NEB</td>
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<td>Urea</td>
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<td>Fisher</td>
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<td>Qiagen</td>
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<td>SK-MEL-31 cells</td>
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<td>MEM</td>
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<td>TrypLE Express</td>
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<td>Gibco</td>
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<td>T4 DNA Ligase</td>
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<td>NEB</td>
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<td>Dimethyl Sulfoxide</td>
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<td>Promega</td>
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<td>Sp6 RNA Polymerase</td>
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<td>Promega</td>
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<td>RNase-Free DNase I</td>
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<td>Promega</td>
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<td>RNase Inhibitor</td>
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<td>Promega</td>
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<td>SensiScript Reverse Transcriptase</td>
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<td>Qiagen</td>
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<td>pCR-2.1-TOPO Vector</td>
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<td>Invitrogen</td>
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<td>DH5&alpha; Compotent Cells</td>
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<td>Invitrogen</td>
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<td>Plasmid Mini Kit</td>
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<td>Qiagen</td>
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<td>Vector NTI</td>
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<td>Invitrogen</td>
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<td>T4 Polynucleotide Kinase</td>
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<td>NEB</td>
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<td>&gamma;-32P-ATP</td>
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primer-free aptamer selection using a random dna library

Aptameren zijn zeer gestructureerd oligonucleotiden (DNA of RNA) die kunnen binden aan doelstellingen met affiniteit die vergelijkbaar zijn met antilichamen 1. Ze zijn geïdentificeerd door middel van een in vitro selectie proces genaamd Systematische Evolutie van de liganden door de exponentiële verrijking (SELEX) om een breed scala aan doelen te herkennen, van kleine moleculen aan eiwitten en andere macromoleculen 2-4. Aptameren hebben eigenschappen die zeer geschikt zijn voor in vivo diagnostische en / of therapeutische toepassingen: Naast goede specificiteit en affiniteit, ze gemakkelijk worden gesynthetiseerd, strengere verwerking omstandigheden te overleven, ze zijn slecht immunogeen, en hun relatief geringe omvang kan resulteren in gemakkelijke penetratie van weefsels. Aptameren die zijn geïdentificeerd door middel van het standaard SELEX proces omvatten doorgaans ~ 80 nucleotiden (nt), aangezien zij meestal geselecteerd uit nucleïnezuur bibliotheken met ~ 40 nt lang gerandomiseerde regio&#39;s plus het vaste primer sites van ~ 20 nt aan elke kant. De vaste primer sequenties kan dus bijna omvatten ~ 50% van de bibliotheek sequenties, en daarom kan positief of negatief compromis identificatie van aptameren in het selectieproces 3, hoewel bioinformatica methoden suggereren dat de vaste sequenties niet significant bijdragen aan de aptameer structuur na selectie 5 . Om deze potentiële problemen, zijn primer sequenties geblokkeerd door complementaire oligonucleotiden of de overstap naar verschillende sequenties halverwege tijdens de rondes van SELEX 6, of ze zijn afgesneden tot 6-9 nt 7, 8. Wen en Gray 9 ontwierp een primer-vrije genomische SELEX methode, waarbij de primer sequenties werden volledig verwijderd uit de bibliotheek voor de selectie en werden vervolgens geregenereerd om versterking van de geselecteerde genomische fragmenten mogelijk te maken. Echter, om gebruik van de techniek, een unieke genomische bibliotheek moet worden gebouwd, die beschikt over beperkte diversiteit, en regeneratie na het ronden van selectie is gebaseerd op een lineaire reamplification stap. Als alternatief zijn pogingen om problemen veroorzaakt door vaste primer sequenties met behulp van zeer efficiënte verdeling te omzeilen een ontmoeting gehad met problemen met betrekking tot PCR-amplificatie 10. We hebben een primer-vrij (PF) selectie methode die aanzienlijk vereenvoudigd SELEX procedures en effectief elimineert primer-storingen 11, 12. De protocollen werken op een eenvoudige wijze. De centrale willekeurige gebied van de bibliotheek wordt gezuiverd, zonder overbodige flankerende sequenties en is gebonden aan een geschikt doelwit (bijvoorbeeld om een ​​gezuiverde eiwit of complexe mengsels zoals mobiele lijnen). Dan is de gebonden sequenties worden verkregen, herenigd met flankerende sequenties, en opnieuw versterkt tot geselecteerde sub-bibliotheken te genereren. Als een voorbeeld, hier zijn we geselecteerd aptameren om S100B, een eiwit marker voor melanoom. Bindingstesten liet Kd s in de 10 -7-10 -8 M serie na een paar ronden van selectie, en we laten zien dat de aptameren effectief te functioneren in een sandwich bindend formaat.

Watch this Scientific Journal Video about Primer-Free Aptamer Selection Using A Random DNA Library at JoVE.com

Primer-Free Aptamer Selection Using A Random DNA Library

SELEX protocollen bestaan ​​uit meerdere ronden van selectie, die elk vereisen regeneratie van gebonden liganden, die op hun beurt behoefte aan vaste primer sequenties aan weerszijden van de willekeurige bibliotheek regio&#39;s. Deze vaste primer sequenties kunnen interfereren met de selectieprocedure (vals positieven en negatieven). Hier presenteren wij een primer-vrij protocol.

Primer-Free aptameer Selection behulp van een random DNA-bibliotheek

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

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