DNA (Taq) polymerase, primers,dNTPs,dsDNA (template)
How does site-directed mutagenesis work?
Create a primer with one nucleotide different from template DNA strand
PCR used to create many new copies of the plasmid containing the new site-specific mutation
What is real-time PCR (qPCR)?
Detects and quantifies the presence of certain gene/s in real time, measurements are made during each cycle of polymerization
How does qPCR work?
Use a fluorescent marker/probe/dye (reporter) and a quencher that blocks the reporter; after one cycle, the quencher frees up the reporter which fluoresces and is recorded
How is qPCR used to diagnose CML?
Primers designed to flank the bcr-abl sequence (Philadelphia chromosome)
If present, you will see increasing fluorescence, otherwise, no fluorescence will be measured
How does recombinant DNA technology work?
Cleave two different sequences with the same restriction enzyme creating sticky ends
Anneal DNA fragments and rejoin with DNA ligase
Amplify with PCR
What is the process of cloning?
Cleave both vector (plasmid) and gene of interest with the same restriction enzyme
Insert gene of interest
Recombinant DNA molecules delivered to bacteria
Plasmids replicated or cloned by the DNA synthesizing machinery of the host
Fusion proteins can then be expressed and purified
What is GFP?
A reporter protein that fluoresces green allowing the identification of the location of fusion proteins
Why do plasmids for cloning contain antibacterial resistance genes?
To allow for specific selection of bacteria expressing the plasmid
