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Techniques Flashcards

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Biology 101 flashcards
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
Q

What are the advantages of gene silencing?

A

Sequence can be designed and stability optimised in silico
No harm to cell
Highly potent: Kd of pM

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26
Q

How can the synthetic siRNA be delivered to cells?

A 
Viral hijacking of Toll like receptors
Liposome
Antibodies
Cationic proteins
Nanotechnology
27
Q

What does RT-PCR measure?

A

Fluorescence increases proportional to starting volume of DNA

28
Q

Which fluorescent molecules can be used for PCR?

A

SybrGreen for dsDNA
Taqman
Molecular Beacons
FRET

29
Q

How does taqman work?

A

Probe anneals and is displaced by polymerase to allow fluorescence

30
Q

How do molecular beacons work?

A

Unfolding and annealing releases quencher

31
Q

What length fragments are involved in rtPCR?

A

100bp to minimise extension time

32
Q

How can the fidelity of probe hybridisation be checked?

A

Plotting a melting curve of T against fluorescence. Peak shown be symmetrical

33
Q

What alternatives to reverse-transcriptase RT-PCR can be used to measure mRNA abundance?

A

northern blotting, in situ hybridisation, ribonuclease protection assays

34
Q

Which genes can be used as controls for northern blots?

A

GAPDH
B-actin
MHC class 1
rRNA

35
Q

How do northern blots measure RNA abundance?

A

darkness of band compared to control

36
Q

What characteristics does a northern blot control gene have?

A

expressed in all cells
expressed at the same copy number
unaffected by cellular conditions

37
Q

What additional controls are needed for a northern blot?

A

negative for no DNA (contamination)
No reverse transcriptase
Positive with no reagents/primers

38
Q

What plot is produced to calculate mRNA concentration?

A

log (copy number) against CT using calibrations from dilutions of control

39
Q

What is the more common method of calculating copy number?

A

estimation using n ^(target-reference)-(experiment)

40
Q

How are antibodies generated?

A

Injection of peptide and anticonjuvant into species

41
Q

Which type of antibody are used?

A

monoclonal

42
Q

How can antibodies be used to detect protein?

A

To directly detect protein using labels

Indirectly in multi-layer assays

43
Q

What do immuno dot blots detect?

A

immobilised protein washed with antibody

44
Q

How is non-specific binding removed in Western blots?

A

Washing with milk protein

45
Q

How can proteins be detected?

A 
Immuno dot blots
Western blots
ChIP
Immunoprecipitation
Co-immunoprecipitation
Fusion or replacement with reporter
46
Q

How does immunoprecipitation work?

A

Protein A/G on solid support binds antibody constant region

47
Q

How sensitive is immunoprecipitation?

A

Very- can detect low quantities of protein

48
Q

How can proteins be produced in vitro?

A

Wheat germ cell or reticulocyte lysates

49
Q

Can immunoprecipitation be used to detect PTM?

A

Yes

50
Q

Where are secondary level antibodies produced?

A

Different organisms to the primary antibody

51
Q

How are antibodies added to a cell for immunocytochemistry?

A

Tissue fixed using formaldehyde
membrane permeabilisation
Addition of antibodies

52
Q

How can rate of protein synthesis be measured?

A

Pulse cells with 35S-Met
Chase with normal Met
Take extracts at different times to measure rates of degradation

53
Q

How is protein removed from DNA in immunoprecipitation?

A

Protinase A

54
Q

How can antibodies be conjugated to reporter proteins?

A

Via aminos/thiols/carboxyl groups

55
Q

What timeframe is immunocytochemistry?

A

A snapshot

56
Q

What are the 3 main classes of fluorescent molecules?

A

Xanthene,
Cyanine
Pyrene derivatives

57
Q

What structure do fluorophores have?

A

aromatic or conjugated

58
Q

Can fluorescent proteins be used in vivo?

A

Yes

59
Q

How can wavelength be restricted for fluorescence?

A

Short/long/band pass, dichroic

60
Q

What distance of interactions does FRET measure?

A

2-8nm

61
Q

What colours can GFP be engineered to?

A

Yellow, Blue, Cyan

62
Q

What makes fluorescent colocalisation difficult?

A

different quantum yeilds, half lives, efficiencies

63
Q

What are the problems with fluorescent proteins?

A 
size,
pH sensitive, 
toxicity, 
mistargetting, 
misfolding produces artefacts
64
Q

How can GFP be engineered for nucleic acids?

A

through TALE

spinach aptamers that bind small organics

Genetics (4 decks)

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