PCR

gina_evans0312
Flashcards by gina_evans0312, updated more than 1 year ago
gina_evans0312
Created by gina_evans0312 over 7 years ago
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Year 2 Flashcards on PCR, created by gina_evans0312 on 12/29/2013.

Resource summary

Question Answer
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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