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Techniques Flashcards

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1
Q

What can site directed mutagenesis be used for?

A

Engineering new proteins with optimal pH, temp, Km, Kcat
Studies of structure/function of proteins
Studies of regulatory DNA sequences

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2
Q

Why was there low recovery in site directed mutagenesis of M13?

A

Poor ligation, strand displacement and mismatch repair

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3
Q

What was the increase in percentage recovery?

A

0.1 to 50%

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4
Q

How can template DNA be removed?

A

Dpn1 digestion of methylated parent DNA
Use of uracil containing DNA
Incorporation of phosphorothiates
Antibiotic selection

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5
Q

What primers are used for QuikChange?

A

2 primers of 25-40nt with central mutation
Tm >78’C
No phosphorylated primers to prevent ligation

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6
Q

Which polymerase is used during QuikChange?

A

KOD or Pfu (lower efficiency)

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7
Q

How are parent strands amplified in QuikChange?

A

PCR,
heat to denature and reanneal parent strands
16-30 cycles

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8
Q

How many colonies are screened following QuikChange?

A

4-6 colonies

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9
Q

Give an example of sequence addition

A

DsbAss signal sequence

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10
Q

Give an example of sequence deletion

A

Amber stop codons which can be read as “stop” or “Gln” in different strains of Ecoli

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11
Q

How can different sequences be inserted during site directed mutagenesis?

A

Cassette
PCR
Sticky feet

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12
Q

How does Cassette mutagenesis work?

A

100nt oligonuclotides are synthesised and incorporated by restriction sites

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13
Q

What length of fragment can be produced by PCR mutagenesis?

A

60nt

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14
Q

What length of insert can be inserted by sticky feet?

A

100nt to several kb

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15
Q

What type of DNA is used for sticky feet mutagenesis?

A

ssDNA of M13 or phagemids

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16
Q

How does saturation mutagenesis work?

A

Random codons synthesised from trimer phosphoramidites with predetermined ratios
Replace codon to determine function

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17
Q

An example of saturation mutagenesis?

A

Galactose oxidase cannot be crystallised with substrate but mutagenesis determines W290 is involved in catalytic and substrate binding

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18
Q

How can nucleic acids be administered to a cell to cause silencing?

A

Addition of RNA encoded as small hairpins in vectors hijacks miRISC pathway

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19
Q

What can gene knockout be used for?

A

Study of gene function
Study of protein function
Therapeutics

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20
Q

Is gene knockout by siRNA permanent?

A

No. Depletes to 20% function

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21
Q

How can mRNA be degraded in vivo?

A

3’UTR forms secondary structures and stimulates deadenylation
miRISC stalls translation for storage in P body
RISC pathway cleaves mRNA for degradation

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22
Q

How does RISC work?

A

dsRNA cleaved by DICER using ATP to 19-21nt in P body
Helicase unwinds using ATP
3’ 2nt overhang binds to PAZ domain of Argonaut exonuclease 3 to coordinate mRNA
PIWI domain cleaves

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23
Q

What size fragments of the hairpin dsRNA are produced for miRISC?

A

21-23nt

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24
Q

What are the problems with gene silencing?

A

Treated as viral genes, hijacking Toll like receptors to cause immune response,
Some cytotoxicity in non-target cells

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25
What are the advantages of gene silencing?
Sequence can be designed and stability optimised in silico No harm to cell Highly potent: Kd of pM
26
How can the synthetic siRNA be delivered to cells?
``` Viral hijacking of Toll like receptors Liposome Antibodies Cationic proteins Nanotechnology ```
27
What does RT-PCR measure?
Fluorescence increases proportional to starting volume of DNA
28
Which fluorescent molecules can be used for PCR?
SybrGreen for dsDNA Taqman Molecular Beacons FRET
29
How does taqman work?
Probe anneals and is displaced by polymerase to allow fluorescence
30
How do molecular beacons work?
Unfolding and annealing releases quencher
31
What length fragments are involved in rtPCR?
100bp to minimise extension time
32
How can the fidelity of probe hybridisation be checked?
Plotting a melting curve of T against fluorescence. Peak shown be symmetrical
33
What alternatives to reverse-transcriptase RT-PCR can be used to measure mRNA abundance?
northern blotting, in situ hybridisation, ribonuclease protection assays
34
Which genes can be used as controls for northern blots?
GAPDH B-actin MHC class 1 rRNA
35
How do northern blots measure RNA abundance?
darkness of band compared to control
36
What characteristics does a northern blot control gene have?
expressed in all cells expressed at the same copy number unaffected by cellular conditions
37
What additional controls are needed for a northern blot?
negative for no DNA (contamination) No reverse transcriptase Positive with no reagents/primers
38
What plot is produced to calculate mRNA concentration?
log (copy number) against CT using calibrations from dilutions of control
39
What is the more common method of calculating copy number?
estimation using n ^(target-reference)-(experiment)
40
How are antibodies generated?
Injection of peptide and anticonjuvant into species
41
Which type of antibody are used?
monoclonal
42
How can antibodies be used to detect protein?
To directly detect protein using labels | Indirectly in multi-layer assays
43
What do immuno dot blots detect?
immobilised protein washed with antibody
44
How is non-specific binding removed in Western blots?
Washing with milk protein
45
How can proteins be detected?
``` Immuno dot blots Western blots ChIP Immunoprecipitation Co-immunoprecipitation Fusion or replacement with reporter ```
46
How does immunoprecipitation work?
Protein A/G on solid support binds antibody constant region
47
How sensitive is immunoprecipitation?
Very- can detect low quantities of protein
48
How can proteins be produced in vitro?
Wheat germ cell or reticulocyte lysates
49
Can immunoprecipitation be used to detect PTM?
Yes
50
Where are secondary level antibodies produced?
Different organisms to the primary antibody
51
How are antibodies added to a cell for immunocytochemistry?
Tissue fixed using formaldehyde membrane permeabilisation Addition of antibodies
52
How can rate of protein synthesis be measured?
Pulse cells with 35S-Met Chase with normal Met Take extracts at different times to measure rates of degradation
53
How is protein removed from DNA in immunoprecipitation?
Protinase A
54
How can antibodies be conjugated to reporter proteins?
Via aminos/thiols/carboxyl groups
55
What timeframe is immunocytochemistry?
A snapshot
56
What are the 3 main classes of fluorescent molecules?
Xanthene, Cyanine Pyrene derivatives
57
What structure do fluorophores have?
aromatic or conjugated
58
Can fluorescent proteins be used in vivo?
Yes
59
How can wavelength be restricted for fluorescence?
Short/long/band pass, dichroic
60
What distance of interactions does FRET measure?
2-8nm
61
What colours can GFP be engineered to?
Yellow, Blue, Cyan
62
What makes fluorescent colocalisation difficult?
different quantum yeilds, half lives, efficiencies
63
What are the problems with fluorescent proteins?
``` size, pH sensitive, toxicity, mistargetting, misfolding produces artefacts ```
64
How can GFP be engineered for nucleic acids?
through TALE | spinach aptamers that bind small organics

Genetics (4 decks)

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