MCB110L_lecture18

MCB110L_lecture18 - MCB110L Lecture 18 03/18/11 Andreas...

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MCB110L Lecture 18 03/18/11 Andreas Martin
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Outline: RT-PCR RNA/DNA quantification Northern blotting Microarrays Solexa sequencing
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retroviral reverse transcriptases with 3 activities: 1) RNA-dependent DNA polymerase -> synthesis of RNA:cDNA hybrid 2) RNaseH -> degradation of RNA in RNA:cDNA hybrid 3) DNA-dependent DNA polymerase -> completion of the ds cDNA For reverse transcription in vitro: first 2 activities are utilized to produce ss cDNA amplification by PCR (using DNA Pol) reverse primer (590 C-term) RT-PCR in class : Qiagen OneStep RT-PCR kit (Omniscript and Sensiscript RT mix, HotStarTaq DNA Pol.) 15 min 95 °C (activation HotStarTaq) 1‘ 94°C; 0.5’ 55°C; 2’ 70°C; 10’ 70°C 30 min 50°C 25x
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RT-PCR amplification to quantify RNA semi-quantify specific mRNA levels by: comparing to a transcript of a control, house-keeping gene (e.g. actin, GAPDH) comparing to an exogenous PCR template of known concentration incorporation of special nucleotides during PCR (DIG-U), detection/quantification using specific antibody more quantitative methods using fluorescent dyes (SYBR- Green), FRET, and molecular beacons (fluorophore/quencher) target molecular beacon hybrid fluorophore quencher
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RT-PCR amplification to quantify RNA Ideal amplification by PCR: Each round doubles the amount of target DNA [DNA] final = [DNA] start x 2 n (n is the number of PCR cycles) In reality: [DNA] final = [DNA] start x ~ 1.9 n (n is the number of PCR cycles) Why does this happen? Innis MA and Gelfand DH (1990). Optimization of PCRs. pp. 3-12 in PCR Protocols (Innis, Gelfand, Sninsky and White, eds.) Academic Press, New York. Important considerations:
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Real-time detection of PCR kinetics to quantify template levels (DNA or RNA) LightCycler (Roche, Idaho Technology) kinetic rather than endpoint approach very rapid (amplification + detection in ~ 20 min) reaction and detection in the same vessel
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quantification of PCR product by FRET or dye binding/intercalation DNA melting curve analysis (proof of DNA identity, mutation analysis) 0 copies 1 copy 10 copies 100 copies 1,000 copies 10,000 copies 100,000 copies 1,000,000 copies Fluorescence Cycle Number accumulation of double-stranded DNA detected with intercalating SYBR Green I dye hybridization probes format uses FRET between 2 probes that come together only when binding to PCR product Real-time detection of PCR kinetics to quantify template levels (DNA or RNA) template levels:
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Northern Blots Key advantage: No need to amplify sequence Key disadvantage: May need to prepare larger amounts of RNA (can’t get down to very small samples or even the single-cell level) General Procedure: o separation of RNA sample by gel (acrylamide or agarose) o transfer of RNA from gel to positively charged membrane (nylon) o detection with a specific hybridization probe Used to detect specific RNAs or monitor expression levels paper nylon membrane gel porous pads
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MCB110L_lecture18 - MCB110L Lecture 18 03/18/11 Andreas...

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