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10 Cards in this Set

  • Front
  • Back
Amplification Strategies
a. PCR (give 2)
c.TMA (give 2)
a.PCR : -Taq polymerase
-Cycling temperatures
c.TMA:- RT, RNase H, and T7 polymerase
- Isothermal reaction
Detection Strategies
1.Which one use intercalator dyes?
2.Prob Chemistries (give 3)
1. SYBR green
2.Probe Chemistries:
Taqman, FRET, Molecular Beacon
What are the advantages of SYBR Green?
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
What are the DISadvantages of SYBR green assay
Detect accumulation of both specific and nonspecific PCR products, thus resulting in a lower specifity than a probe-assay.
Give characteristics of the following fluorescent probes:
2.Molecular Beacon
-Fluorescent Resonance Energy Transfer
1.Taqman: One-doubly labeled probe
2.Molecular Beacon: One doubly-labeled probe with hairpin molecule.
Intact Taqman Probe
Proximity of REPORTER fluorophore to QUENCHER fluorophore results in ______________________
suppression of the Reporter during excitation.
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
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
What are the advantages to real time PCR
-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.
What are the disadvantages of real time PCR
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.