Questão 1
Questão
What is site directed mutation?
Responda
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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
Questão 2
Questão
What form must the DNA be in for Site Directed Mutagenesis?
Questão 3
Questão
The primer for site directed mutagenesis is designed to have one or more mismatches
Questão 4
Questão
Once the mismatch has been achieved, how is it used?
Responda
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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
Questão 5
Questão
What is specific about the DNA used in the Kunkel Method?
Questão 6
Questão
What is Incorporated into the original DNA in order to make it susceptible to degrading
Responda
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Uracil
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Methyl-cytosine
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Thymidine-3-phosphate
Questão 7
Questão
Like with site directed, the primer annealed to the DNA is designed to create mismatches
Questão 8
Questão
In both site directed and the Kunkel method, how is the primer extended?
Responda
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DNA polymerase
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Taq Polymerase
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RNA polymerase
Questão 9
Questão
Once the DNA has been inserted, what happens to the original DNA strand?
Responda
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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
Questão 10
Questão
The partially degraded strand is them re-synthesised by DNA repair proteins, finalising the mutation
Questão 11
Questão
Which method has a way of determining which plasmid has the mutation?
Responda
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Site- Directed
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Altered Site
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Kunkel Method
Questão 12
Questão
What is particular about the plasmid used in the Altered Sites method?
Questão 13
Questão
What oligonucleotide primers are used during Altered Site mutagenisis?
Questão 14
Questão
What is the result, after DNA replication has occurred?
Responda
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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
Questão 15
Questão
What causes the mutagenesis in the 'Quickchange' PCR method?
Responda
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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
Questão 16
Questão
Why is Dpn used to degrade the original DNA?
Responda
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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
Questão 17
Questão
Site directed mutagenesis is unique in that no structural knowledge of the protein is required
Questão 18
Questão
State the two methods by which non-directed mutation occurs in evolution
Responda
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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
Questão 19
Questão
These random mutations can be mimicked in a lab
Questão 20
Questão
Random mutagenesis works best by introducing many mutations at the same time
Questão 21
Questão
What will happen when this DNA replicates?
Questão 22
Questão
Primers can be more specific and limit the choice of potential aa further
Questão 23
Questão
Name an Error prone DNA polymerase
Questão 24
Questão
Name the two conditions necessary for Error Prone mutagensis
Questão 25
Questão
DNA shuffling is used to create fusion proteins from two unrelated genes
Questão 26
Questão
Why does gene shuffling work?
Responda
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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
Questão 27
Questão
What protein is used to randomly cut up two related genes for DNA shuffling?
Responda
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DNAase I
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DNAase III
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EcoRV
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EcoRI
Questão 28
Questão
The randomly cut up sequences are then ligated back together
Questão 29
Questão
When creating hybrids, the traditional approach of screening colonies for hybrids is not efficient- so we use robots
Questão 30
Questão
Name the two main methods of analysing mutants quickly
Questão 31
Questão
The advantage of Protein Surface Display in improved mutations is?
Responda
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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
Questão 32
Questão
Once the variants are bound, they are washed so strongly only powerfully binding variants remain bound
Questão 33
Questão
Once the most improved variation has been identified, what can happen to it?
Questão 34
Questão
Chemical Compartmentalisation uses an oil/acetyl emulsion
Questão 35
Questão
Why are the droplets important?
Responda
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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
Questão 36
Questão
The DNA cannot be transcribed or translated in the droplets, only sequenced
Questão 37
Questão
Once the protein has been made, how are they assayed?
Questão 38
Questão
How is fluorescence used for separation in Fluorescent Activated Cell Sorters?
Responda
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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