PCR

Frage	Antworten
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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