What is site directed mutation?
Where mutagenesis is directed towards a specific site
Where certain sites are more prone to mutagenesis than others
Where certain sites cannot be mutated and must be worked around
What form must the DNA be in for Site Directed Mutagenesis?
ssRNA
ssDNA
dsDNA
The primer for site directed mutagenesis is designed to have one or more mismatches
Once the mismatch has been achieved, how is it used?
During repair, the old base will be replaced with a new match
During replication, half the daughter cells will carry the mutation
What is specific about the DNA used in the Kunkel Method?
It's dsDNA
It's ssDNA
From the Slo + form of E.Coli
From the dut ung. form of E.Coli
What is Incorporated into the original DNA in order to make it susceptible to degrading
Uracil
Methyl-cytosine
Thymidine-3-phosphate
Like with site directed, the primer annealed to the DNA is designed to create mismatches
In both site directed and the Kunkel method, how is the primer extended?
DNA polymerase
Taq Polymerase
RNA polymerase
Once the DNA has been inserted, what happens to the original DNA strand?
The Uracil cause it to be partially degraded
The Methyl-cytosine cause it to be partially degraded
The Thymidine-3-Phosphate cause it to be partially degraded
Being removed alone with the bp next to them
The partially degraded strand is them re-synthesised by DNA repair proteins, finalising the mutation
Which method has a way of determining which plasmid has the mutation?
Site- Directed
Altered Site
Kunkel Method
What is particular about the plasmid used in the Altered Sites method?
It contains a non-functional Amp resistance gene
It contains a gene for DNA repair proteins
It's dsRNA
What oligonucleotide primers are used during Altered Site mutagenisis?
Mutant primer
Ampicillin 'Repair' primer
Ampicillin Non-Function primer
What is the result, after DNA replication has occurred?
The desired plasmid has the mutation, and tetracyline and ampicillin resistance
The desired plasmid has the mutation and tet resistance but no ampicillin resistance
The desired plasmid has the mutation and ampicillin resistance but no tetracyline resistance
What causes the mutagenesis in the 'Quickchange' PCR method?
A two primers containing the mutation
Rapid replication of the Site Directed mutant
One primer contains a mutation, the other is used to create the mutated plasmid
The original DNA is then degraded, leaving only the mutated plasmid
Why is Dpn used to degrade the original DNA?
It degrades only methylated DNA
It degrades only acetylated DNA
It degrades only ibiquitinated DNA
Site directed mutagenesis is unique in that no structural knowledge of the protein is required
State the two methods by which non-directed mutation occurs in evolution
Gradual accumulation of beneficiary mutations
Gradual loss of unnecessary DNA
Recombination of genes to form hybrid protein
These random mutations can be mimicked in a lab
Random mutagenesis works best by introducing many mutations at the same time
What will happen when this DNA replicates?
The amino acid marked by the N's will be randomised
The Leu will be deleted
The N's are a stop codon, halting the gene
Primers can be more specific and limit the choice of potential aa further
Name an Error prone DNA polymerase
Taq
T7
Pol
Name the two conditions necessary for Error Prone mutagensis
An error prone polymerase
Conditions designed to increase error
No DNA repair enzymes
Restriction enzymes
DNA shuffling is used to create fusion proteins from two unrelated genes
Why does gene shuffling work?
Ligation repairs DNA errors
Related genes have overlapping areas
Related genes can ligate together with less error
What protein is used to randomly cut up two related genes for DNA shuffling?
DNAase I
DNAase III
EcoRV
EcoRI
The randomly cut up sequences are then ligated back together
When creating hybrids, the traditional approach of screening colonies for hybrids is not efficient- so we use robots
Name the two main methods of analysing mutants quickly
Surface display of proteins
Elution
Chemical compartmentalisation
The advantage of Protein Surface Display in improved mutations is?
Improved mutations will bind more strongly to immobalised substrate
They react more strongly to dyes
They don't degrade as easily
Once the variants are bound, they are washed so strongly only powerfully binding variants remain bound
Once the most improved variation has been identified, what can happen to it?
It's washed off and sequenced
It's washed off and improved again
Chemical Compartmentalisation uses an oil/acetyl emulsion
Why are the droplets important?
They make reading the DNA easier
Each droplet contains an individual mutation
Each droplet contains mutants that compliment each other
The DNA cannot be transcribed or translated in the droplets, only sequenced
Once the protein has been made, how are they assayed?
Robots put them on multi-well dishes for assay
Fluorescent activated cell sorters are used
How is fluorescence used for separation in Fluorescent Activated Cell Sorters?
The more fluorescent product is produced, the better the enzyme
The stronger the enzyme-substrate binding, the better the enzyme
The more fluorescent substrate is used, the better the enzyme
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