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 arbeidet ble støttet med tilskudd fra Pittsburgh Foundation og National Institutes of Health (NIH) [GM087798, CA148629, ES019498] til RWS. Støtte for UPCI Lentiviral Facility ble gitt til RWS ved Cancer Center Support Grant fra National Institutes of Health [P30 CA047904]. Støtte ble også gitt av Universitetet i Pittsburgh Institutt for farmakologi og Chemical Biology til DS. Støtte ble også gitt av ESTRO til CV.

1. Molekylær Beacon Design 1.1 Utforming og bestiller din molekylære fyrtårn Utformingen av molekylære fyrtårn må vurdere følgende: <ol class="lalpha"><li> Vi har gode resultater med 43-mer DNA oligonukleotider. Den GC innhold bør være&gt; 32%. Ekskludere en G base ved 5 &#39;enden. </li><li> Be om 6-FAM (6-Carboxyfluorescein) konjugering på 5 &#39;enden og Dabcyl som en 3&#39; modifikasjon. </li><li> Plasseringen av lesjonen bør være på basen # 6 fra...

<ol>
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Det er over 600 siteringer bruker begrepet &quot;molekylære fyrtårn&quot; for å påvise og kvantifisere mange molekyler og enzymatiske aktiviteter, inkludert helicase aktivitet 6, DNA polymerase aktivitet 7,8, DNA ligase aktivitet 9, telomerase aktivitet 10, DNA photolyase aktivitet 11 og DNA / RNA 12 hybrider, blant mange andre. I tillegg til bruk av molekylære signaler for måling av DNA-reparasjon som er oppført ovenfor, har vi og andre demonstrer...

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

Vi beskriver en metode for kvantitativ, real-time måling av DNA glykosylase og AP endonuclease aktiviteter i celle kjernefysiske lysates med Base excision reparasjon (BER) molekylære beacons. Underlaget (beacon) består av en deoxyoligonucleotide inneholder en enkelt base lesjon med en 6-Carboxyfluorescein (6-FAM) moiety konjugert til 5&#39;end og en Dabcyl moiety konjugert til 3 &#39;enden av oligonukleotid. Den BER molekylære fyrtårn er 43 baser i lengde og sekvensen er utformet for å fremme dannelsen av en stilk-sløyfe struktur med 13 nukleotider i loopen og 15 basepar i stammen 1,2. Når brettet i denne konfigurasjonen den 6-FAM moiety er slukket ved Dabcyl i en ikke-fluorescerende måte via Förster Resonance Energy Transfer (FRET) 3,4. Lesjonen er plassert slik at følgende basen lesjon fjerning og strand scission de resterende fem basen oligonukleotid inneholder 6-FAM moiety frigjøres fra stammen. Slipp ennd løsrivelse fra slukkeren (Dabcyl) resulterer i en økning av fluorescens som står i forhold til nivået av DNA-reparasjon. Ved å samle flere leser av fluorescens verdier, er sanntids vurdering av BER aktivitet mulig. Bruken av standard kvantitativ real-time PCR instrumenter tillater samtidig analyse av en rekke prøver. Utformingen av disse BER molekylære signaler, med en enkelt base lesjon, er mottagelig for kinetiske analyser, BER kvantifisering og hemmer validering og er tilpasningsdyktig for kvantifisering av DNA Repair aktivitet i vev og tumorcellelinjer lysates eller med renset proteiner. Analysen av BER aktivitet i tumor lysates eller vev aspirates bruker disse molekylære signaler kan være aktuelt å funksjonelle biomarkør målinger. Videre gir analysen av BER aktivitet med renset proteiner ved hjelp av denne kvantitative analysen en rask, high-throughput metode for oppdagelsen og validering av BER hemmere.

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

Vi beskriver en metode for kvantitativ, real-time måling av DNA glykosylase og AP endonuclease aktiviteter i celle kjernefysiske lysates. Analysen gir tallene for DNA Repair aktivitet mottagelig til kinetisk analyse og er tilpasningsdyktig for kvantifisering av DNA Repair aktivitet i vev og tumor lysates eller med rensede proteiner.

Kvantitativ, Real-time analyse av Base Excision Reparasjon aktivitet i Cell lysates utnytte Lesjon-spesifikke 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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