Pregunta 1
Pregunta
What is site directed mutation?
Respuesta
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Where mutagenesis is directed towards a specific site
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Where certain sites are more prone to mutagenesis than others
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Where certain sites cannot be mutated and must be worked around
Pregunta 2
Pregunta
What form must the DNA be in for Site Directed Mutagenesis?
Pregunta 3
Pregunta
The primer for site directed mutagenesis is designed to have one or more mismatches
Pregunta 4
Pregunta
Once the mismatch has been achieved, how is it used?
Respuesta
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During repair, the old base will be replaced with a new match
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During replication, half the daughter cells will carry the mutation
Pregunta 5
Pregunta
What is specific about the DNA used in the Kunkel Method?
Pregunta 6
Pregunta
What is Incorporated into the original DNA in order to make it susceptible to degrading
Respuesta
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Uracil
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Methyl-cytosine
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Thymidine-3-phosphate
Pregunta 7
Pregunta
Like with site directed, the primer annealed to the DNA is designed to create mismatches
Pregunta 8
Pregunta
In both site directed and the Kunkel method, how is the primer extended?
Respuesta
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DNA polymerase
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Taq Polymerase
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RNA polymerase
Pregunta 9
Pregunta
Once the DNA has been inserted, what happens to the original DNA strand?
Respuesta
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The Uracil cause it to be partially degraded
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The Methyl-cytosine cause it to be partially degraded
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The Thymidine-3-Phosphate cause it to be partially degraded
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Being removed alone with the bp next to them
Pregunta 10
Pregunta
The partially degraded strand is them re-synthesised by DNA repair proteins, finalising the mutation
Pregunta 11
Pregunta
Which method has a way of determining which plasmid has the mutation?
Respuesta
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Site- Directed
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Altered Site
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Kunkel Method
Pregunta 12
Pregunta
What is particular about the plasmid used in the Altered Sites method?
Pregunta 13
Pregunta
What oligonucleotide primers are used during Altered Site mutagenisis?
Pregunta 14
Pregunta
What is the result, after DNA replication has occurred?
Respuesta
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The desired plasmid has the mutation, and tetracyline and ampicillin resistance
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The desired plasmid has the mutation and tet resistance but no ampicillin resistance
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The desired plasmid has the mutation and ampicillin resistance but no tetracyline resistance
Pregunta 15
Pregunta
What causes the mutagenesis in the 'Quickchange' PCR method?
Respuesta
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A two primers containing the mutation
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Rapid replication of the Site Directed mutant
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One primer contains a mutation, the other is used to create the mutated plasmid
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The original DNA is then degraded, leaving only the mutated plasmid
Pregunta 16
Pregunta
Why is Dpn used to degrade the original DNA?
Respuesta
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It degrades only methylated DNA
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It degrades only acetylated DNA
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It degrades only ibiquitinated DNA
Pregunta 17
Pregunta
Site directed mutagenesis is unique in that no structural knowledge of the protein is required
Pregunta 18
Pregunta
State the two methods by which non-directed mutation occurs in evolution
Respuesta
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Gradual accumulation of beneficiary mutations
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Gradual loss of unnecessary DNA
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Recombination of genes to form hybrid protein
Pregunta 19
Pregunta
These random mutations can be mimicked in a lab
Pregunta 20
Pregunta
Random mutagenesis works best by introducing many mutations at the same time
Pregunta 21
Pregunta
What will happen when this DNA replicates?
Pregunta 22
Pregunta
Primers can be more specific and limit the choice of potential aa further
Pregunta 23
Pregunta
Name an Error prone DNA polymerase
Pregunta 24
Pregunta
Name the two conditions necessary for Error Prone mutagensis
Pregunta 25
Pregunta
DNA shuffling is used to create fusion proteins from two unrelated genes
Pregunta 26
Pregunta
Why does gene shuffling work?
Respuesta
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Ligation repairs DNA errors
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Related genes have overlapping areas
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Related genes can ligate together with less error
Pregunta 27
Pregunta
What protein is used to randomly cut up two related genes for DNA shuffling?
Respuesta
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DNAase I
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DNAase III
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EcoRV
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EcoRI
Pregunta 28
Pregunta
The randomly cut up sequences are then ligated back together
Pregunta 29
Pregunta
When creating hybrids, the traditional approach of screening colonies for hybrids is not efficient- so we use robots
Pregunta 30
Pregunta
Name the two main methods of analysing mutants quickly
Pregunta 31
Pregunta
The advantage of Protein Surface Display in improved mutations is?
Respuesta
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Improved mutations will bind more strongly to immobalised substrate
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They react more strongly to dyes
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They don't degrade as easily
Pregunta 32
Pregunta
Once the variants are bound, they are washed so strongly only powerfully binding variants remain bound
Pregunta 33
Pregunta
Once the most improved variation has been identified, what can happen to it?
Pregunta 34
Pregunta
Chemical Compartmentalisation uses an oil/acetyl emulsion
Pregunta 35
Pregunta
Why are the droplets important?
Respuesta
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They make reading the DNA easier
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Each droplet contains an individual mutation
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Each droplet contains mutants that compliment each other
Pregunta 36
Pregunta
The DNA cannot be transcribed or translated in the droplets, only sequenced
Pregunta 37
Pregunta
Once the protein has been made, how are they assayed?
Pregunta 38
Pregunta
How is fluorescence used for separation in Fluorescent Activated Cell Sorters?
Respuesta
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The more fluorescent product is produced, the better the enzyme
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The stronger the enzyme-substrate binding, the better the enzyme
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The more fluorescent substrate is used, the better the enzyme