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10 Cards in this Set
- Front
- Back
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Amplification Strategies
a. PCR (give 2) b.NASBA c.TMA (give 2) |
a.PCR : -Taq polymerase
-Cycling temperatures b.NASBA c.TMA:- RT, RNase H, and T7 polymerase - Isothermal reaction |
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Detection Strategies
1.Which one use intercalator dyes? 2.Prob Chemistries (give 3) |
1. SYBR green
2.Probe Chemistries: Taqman, FRET, Molecular Beacon |
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What are the advantages of SYBR Green?
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Ideally suited for rapid screening
Lower costs than a probe-assay Simple way of monitoring accumulating amplicon Couple with melting curve (Tm) analysis to increase assay specifity |
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What are the DISadvantages of SYBR green assay
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Detect accumulation of both specific and nonspecific PCR products, thus resulting in a lower specifity than a probe-assay.
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Give characteristics of the following fluorescent probes:
1.Taqman 2.Molecular Beacon 3.FRET -Fluorescent Resonance Energy Transfer |
1.Taqman: One-doubly labeled probe
2.Molecular Beacon: One doubly-labeled probe with hairpin molecule. 3.FRET: |
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Intact Taqman Probe
Proximity of REPORTER fluorophore to QUENCHER fluorophore results in ______________________ |
suppression of the Reporter during excitation.
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Cleaved Taqman Probe
5' nuclease activity of Taq cleaves Reporter dye, which is now no longer under influence of Quencher -Probe cleavage occurs only if ________________________ |
probe is hybridized to its target
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MGB probes and Black Hole Quenchers
1.DNA______ ligand is congugated to DNA probe 2. MGB enhances ___ resulting in shorter probes 3. Pcr rxn can be run at _____ temperatures 4._______ probes provide better dicrimination evn with A/T rich sequences 5.____ and ____ can be used instead of another fluorescent dye. |
1.Minor groove landing
2. Tm of the probe 3.higher temperatures 4. shorter probes 5.BHQ or Dark Quenchers |
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What are the advantages to real time PCR
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-Rapid turn around time.
-Ease of performing quantitative assays. -Ability to perform a multiplex assay. -Reduce risk of contamination using a closed system. -High level of assay specificity when using a probe -Post PCR processing is eliminated, which reduces labor and material cost. |
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What are the disadvantages of real time PCR
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1.Requires the synthesis of different of different target
2.Stringent rules for primer and probe designs= conversion from conventional to real time assay may require modification c. Royalty payments for additional patents. |
