Techniques Flashcards
What can site directed mutagenesis be used for?
Engineering new proteins with optimal pH, temp, Km, Kcat
Studies of structure/function of proteins
Studies of regulatory DNA sequences
Why was there low recovery in site directed mutagenesis of M13?
Poor ligation, strand displacement and mismatch repair
What was the increase in percentage recovery?
0.1 to 50%
How can template DNA be removed?
Dpn1 digestion of methylated parent DNA
Use of uracil containing DNA
Incorporation of phosphorothiates
Antibiotic selection
What primers are used for QuikChange?
2 primers of 25-40nt with central mutation
Tm >78’C
No phosphorylated primers to prevent ligation
Which polymerase is used during QuikChange?
KOD or Pfu (lower efficiency)
How are parent strands amplified in QuikChange?
PCR,
heat to denature and reanneal parent strands
16-30 cycles
How many colonies are screened following QuikChange?
4-6 colonies
Give an example of sequence addition
DsbAss signal sequence
Give an example of sequence deletion
Amber stop codons which can be read as “stop” or “Gln” in different strains of Ecoli
How can different sequences be inserted during site directed mutagenesis?
Cassette
PCR
Sticky feet
How does Cassette mutagenesis work?
100nt oligonuclotides are synthesised and incorporated by restriction sites
What length of fragment can be produced by PCR mutagenesis?
60nt
What length of insert can be inserted by sticky feet?
100nt to several kb
What type of DNA is used for sticky feet mutagenesis?
ssDNA of M13 or phagemids
How does saturation mutagenesis work?
Random codons synthesised from trimer phosphoramidites with predetermined ratios
Replace codon to determine function
An example of saturation mutagenesis?
Galactose oxidase cannot be crystallised with substrate but mutagenesis determines W290 is involved in catalytic and substrate binding
How can nucleic acids be administered to a cell to cause silencing?
Addition of RNA encoded as small hairpins in vectors hijacks miRISC pathway
What can gene knockout be used for?
Study of gene function
Study of protein function
Therapeutics
Is gene knockout by siRNA permanent?
No. Depletes to 20% function
How can mRNA be degraded in vivo?
3’UTR forms secondary structures and stimulates deadenylation
miRISC stalls translation for storage in P body
RISC pathway cleaves mRNA for degradation
How does RISC work?
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
What size fragments of the hairpin dsRNA are produced for miRISC?
21-23nt
What are the problems with gene silencing?
Treated as viral genes, hijacking Toll like receptors to cause immune response,
Some cytotoxicity in non-target cells
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
How can the synthetic siRNA be delivered to cells?
Viral hijacking of Toll like receptors Liposome Antibodies Cationic proteins Nanotechnology
What does RT-PCR measure?
Fluorescence increases proportional to starting volume of DNA
Which fluorescent molecules can be used for PCR?
SybrGreen for dsDNA
Taqman
Molecular Beacons
FRET
How does taqman work?
Probe anneals and is displaced by polymerase to allow fluorescence
How do molecular beacons work?
Unfolding and annealing releases quencher
What length fragments are involved in rtPCR?
100bp to minimise extension time
How can the fidelity of probe hybridisation be checked?
Plotting a melting curve of T against fluorescence. Peak shown be symmetrical
What alternatives to reverse-transcriptase RT-PCR can be used to measure mRNA abundance?
northern blotting, in situ hybridisation, ribonuclease protection assays
Which genes can be used as controls for northern blots?
GAPDH
B-actin
MHC class 1
rRNA
How do northern blots measure RNA abundance?
darkness of band compared to control
What characteristics does a northern blot control gene have?
expressed in all cells
expressed at the same copy number
unaffected by cellular conditions
What additional controls are needed for a northern blot?
negative for no DNA (contamination)
No reverse transcriptase
Positive with no reagents/primers
What plot is produced to calculate mRNA concentration?
log (copy number) against CT using calibrations from dilutions of control
What is the more common method of calculating copy number?
estimation using n ^(target-reference)-(experiment)
How are antibodies generated?
Injection of peptide and anticonjuvant into species
Which type of antibody are used?
monoclonal
How can antibodies be used to detect protein?
To directly detect protein using labels
Indirectly in multi-layer assays
What do immuno dot blots detect?
immobilised protein washed with antibody
How is non-specific binding removed in Western blots?
Washing with milk protein
How can proteins be detected?
Immuno dot blots Western blots ChIP Immunoprecipitation Co-immunoprecipitation Fusion or replacement with reporter
How does immunoprecipitation work?
Protein A/G on solid support binds antibody constant region
How sensitive is immunoprecipitation?
Very- can detect low quantities of protein
How can proteins be produced in vitro?
Wheat germ cell or reticulocyte lysates
Can immunoprecipitation be used to detect PTM?
Yes
Where are secondary level antibodies produced?
Different organisms to the primary antibody
How are antibodies added to a cell for immunocytochemistry?
Tissue fixed using formaldehyde
membrane permeabilisation
Addition of antibodies
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
How is protein removed from DNA in immunoprecipitation?
Protinase A
How can antibodies be conjugated to reporter proteins?
Via aminos/thiols/carboxyl groups
What timeframe is immunocytochemistry?
A snapshot
What are the 3 main classes of fluorescent molecules?
Xanthene,
Cyanine
Pyrene derivatives
What structure do fluorophores have?
aromatic or conjugated
Can fluorescent proteins be used in vivo?
Yes
How can wavelength be restricted for fluorescence?
Short/long/band pass, dichroic
What distance of interactions does FRET measure?
2-8nm
What colours can GFP be engineered to?
Yellow, Blue, Cyan
What makes fluorescent colocalisation difficult?
different quantum yeilds, half lives, efficiencies
What are the problems with fluorescent proteins?
size, pH sensitive, toxicity, mistargetting, misfolding produces artefacts
How can GFP be engineered for nucleic acids?
through TALE
spinach aptamers that bind small organics
