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Criado por gina_evans0312
mais de 10 anos atrás
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Questão | Responda |
Anneal Primers | Short DNA sequences that define the area to be amplified |
Nestled primers | Bind inside the first set and are used for specificity |
Melt DNA at... | 95 degrees |
Primers bind after... | Melting |
Anneal at... | 40-60 degrees |
Extend primers at... | 72 degrees |
The stages are repeated... | 20-30 times |
The ends of the amplified DNA represent | The primers, not the actual DNA |
DNA Polymerases Vary In | Error Rate, Thermostability and DNA length required |
Uses of PCR- Viruses | Diagnosis of Viral Infections |
Uses- Cloning | Creates millions of copies of a sequence very quickly |
Uses- DNA | Indetification/ characterisation of a sequence |
Quirk of Taq Polymerase | A residues are added to the end of sequences |
T- Vectors | Add T residues to the ends to be ligated, which are complimentary to the A's left by Taq |
TOPO Isomerase | An enzyme conjugated to the T/A residues, giving the vector a built in ligase |
Introduction of a Restricton Site | Done via the primer- use the site made to make insertion into the vector easier |
Reverse Transcriptase PCR | Convert mRNA to DNA in PCR reaction |
Reverse Transcriptase PCR- Primers | Can be for PolyA tail or gene specific |
Reverse Transcriptase PCR- Tth Thermostable DNA Polymerase | Has both reverse transcriptae and helicase activity and can be used for both steps |
Quantitative PCR | The measuring of product as it is formed to determine how much DNA there was originally |
DNA Production in PCR | Slows rapidly towards the end of the reaction |
Light Cycler Assay Probes | Fluorescent beads that binds during amplification |
Light Cycler Assay- Fluorescence | More DNA produced, more fluorescence detected |
Use of Taqman Assay | Identify amounts of a specific DNA sequence |
Use of Taqman Assay Probes | Contain fluorophore near a quencher |
Use of Taqman Assay Amplification | Amplification of DNA forces quencher of probe, causing fluorophore to fluoresce |
Molecular Beacons | Probe with a quencher and fluorophore- 2ndary strucuture of unbound probe holds them together |
Binding of Molecular Probes | Puts fluorophore & quencher far enough apart that fluorophore fluoresces |
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