Name: quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons
Uploaded: 2012-08-06T13:00:00
Description: Watch this Scientific Journal Video about Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons at JoVE.com

Dette arbejde blev støttet af tilskud fra Pittsburgh Foundation og National Institutes of Health (NIH) [GM087798, CA148629; ES019498] til RWS. Støtte til UPCI lentiviral faciliteten blev stillet til rådighed RWS fra Cancer Center Support Grant fra National Institutes of Health [P30 CA047904]. Der blev også leveret af University of Pittsburgh Institut for Farmakologi &amp; Chemical Biology til DS. Der blev også leveret af ESTRO til CV.

1. Molekylær Beacon Design 1,1 Design og bestilling din molekylær beacon Udformningen af ​​din molekylær fyrtårn skal overveje følgende: <ol class="lalpha"><li> Vi har gode resultater med 43-mer DNA-oligonukleotider. GC-indholdet bør være&gt; 32%. Udelukke G base ved 5&#39;-enden. </li><li> Anmodningen 6-FAM (6-carboxyfluorescein) konjugering af 5&#39;-enden og Dabcyl som 3&#39;modifikation. </li><li> Positionen af ​​læsion bør være på basen # 6...

<ol>
	<li>Mutamba, 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=XRCC1+and+base+excision+repair+balance+in+response+to+nitric+oxide.">XRCC1 and base excision repair balance in response to nitric oxide.</a> DNA Repair (Amst). 10, 1282-1293 (2011).</li><li>Tang, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-methylpurine+DNA+glycosylase+and+DNA+polymerase+beta+modulate+BER+inhibitor+potentiation+of+glioma+cells+to+temozolomide.">N-methylpurine DNA glycosylase and DNA polymerase beta modulate BER inhibitor potentiation of glioma cells to temozolomide.</a> Neuro-oncology. 13, 471-486 (2011).</li><li>Clegg, R. 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Int. Ed. Engl. 41, 3620-3622 (2002).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+molecular+beacon+to+monitor+activity+of+E.+coli+DNA+ligase.">Using molecular beacon to monitor activity of E. coli DNA ligase.</a> The Analyst. 130, 350-357 (2005).</li><li>Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+beacons+for+the+detection+of+DNA+and+telomerase+activity.">Catalytic beacons for the detection of DNA and telomerase activity.</a> J. Am. Chem. Soc. 126, 7430-7431 (2004).</li><li>Kundu, L. M., Burgdorf, L. 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Der er over 600 citater anvender udtrykket &quot;molekylære mærke&quot; til detektion og kvantificering af adskillige molekyler og enzymatiske aktiviteter, herunder helicase-aktivitet 6, DNA-polymerase-aktivitet 7,8, DNA-ligase-aktivitet 9, telomeraseaktivitet 10, DNA photolyase aktivitet 11 og DNA / RNA hybrider 12, blandt mange andre. Ud over anvendelse af Molecular Beacons til måling af DNA-reparation, der er opført ovenfor, har vi og andre viste a...

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quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons

Beskriver vi en fremgangsmåde til kvantitativ real-time måling af DNA-glycosylase og AP-endonuclease aktiviteter cellekerner lysater under anvendelse base excision reparation (BER) Molecular Beacons. Substratet (fyr) består af en deoxyoligonukleotid indeholdende en enkelt base læsion med en 6-carboxyfluorescein (6-FAM)-delen konjugeret til 5&#39;-enden og en Dabcyl del konjugeret til 3&#39;-enden af ​​oligonukleotidet. BER molekylære mærke er 43 baser i længde og sekvens udformet til at fremme dannelsen af en stem-loop-struktur med 13 nukleotider i sløjfen og 15 basepar i stammen 1,2. Når den er foldet i denne konfiguration 6-FAM-delen standses ved Dabcyl i en ikke-fluorescerende måde via Forster Resonance Energy Transfer (FRET) 3,4. Læsionen er anbragt således, at efter grundlæggende læsion fjernelse og strengspaltning resterende fem baser oligonukleotid indeholdende 6-FAM del frigives fra stilken. Frigive ennd frigørelse fra quencher (dabcyl) resulterer i en forøgelse af fluorescens, som er proportional med niveauet af DNA-reparation. Ved at samle flere læser af fluorescens værdier tidstro vurdering af BER aktivitet er mulig. Anvendelse af standard kvantitativ realtids-PCR instrumenter tillader den samtidige analyse af flere prøver. Udformningen af ​​disse BER Molecular Beacons med en enkelt base læsion, er egnet til kinetiske analyser, BER kvantificering og inhibitor validering og kan tilpasses til kvantificering af DNA-reparation aktivitet i væv og tumorer cellelysater eller oprensede proteiner. Analysen af ​​BER aktivitet i tumor lysater eller væv suger ved hjælp af disse molekylære beacons kan gælde for funktionelle biomarkør målinger. Yderligere analyse af BER-aktivitet med oprensede proteiner under anvendelse af denne kvantitative assay tilvejebringer en hurtig, high-throughput fremgangsmåde til opdagelse og validering af BER-inhibitorer.

Watch this Scientific Journal Video about Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons at JoVE.com

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

Beskriver vi en fremgangsmåde til kvantitativ real-time måling af DNA-glycosylase og AP-endonuclease aktiviteter cellekerner lysater. Assayet giver hastigheder af DNA-reparation aktivitet kan underkastes kinetisk analyse og kan tilpasses til kvantificering af DNA-reparation aktivitet i væv og tumorer lysater eller oprensede proteiner.

Kvantitativ, Real-time analyse af base excision reparation aktivitet i cellelysater Udnyttelse Læsionssynligheden-specifikke Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

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