BIOL 466 Study Guide - Final Guide: Protein Synthesis Inhibitor, Helper Virus, Protein Tag
24 Apr 2019
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Ampicillin resistance
- Inhibits peptidoglycan crosslinking
- Bla gene (beta-lactamase) cuts lactam ring of peptidoglycan
Chloramphenicol
- Inhibits peptidyl transferase
- Protein synthesis inhibitor
- Cat gene
Kanamycin
- Aminoglycoside
- Interferes with protein synthesis at ribosome (misreading of mRNA)
- Aph genes
a-Complementation vectors:
- carry the coding sequence for the first 131 aa of the LacZ gene (a) that complement a
defective LacZ protein encoded by the host bacteria (w). Allows easy selection of
colonies that contain a recombinant plasmid.
- pBluescript
- if DNA is inserted, no functional alpha fragment produced, no functional b GAL
Expression Vectors:
- contain strong promoters, in certain cases regulatable, that generate large amounts of
protein encoded by the cloned sequence of DNA. Allows for easy production of protein
(with or without an affinity tag).
Sequencing:
- some plasmids have the origin of replication for single- stranded phage (i.e.: M13).
Produces large quantities of single- stranded template DNA that can then be used for
sequencing DNA (needs a helper phage to drive the process).
Shuttle vectors:
- have two separate origins of replication and two different selectable markers. Allows for
replication in two separate organisms.
EtBR
- intercalates DNA
PCR – whats needed?
- Thermostable DNA pol (Taq *no proof reading* or Pfu)
- dNTPs
- oligonucleotides (primers)
- template DNA
- process
o Denature DNA at 95oC – converts DNA to single stranded DNA and allows oligos
to hybridize. 30sec.-1min.
o Hybridize oligos at 45-55oC – provides a “starting point” for the polymerase,
should be below Tm of oligos (usually Tm-5oC). 30sec.-1min.
o Extend template at 72oC – optimal temperature for the thermostable
polymerases can vary from 68-72oC. Generally 1min./kb
o repeat
Tm
- the temp at which dsDNA melts into ssDNA (50%)
- affected by
o length of oligos,
o G/C content
o Salt conc
o Mg2+ conc (cofactor for DNA pol)
- Calculating TM
o Tm=2X(A+T) + 4X(G+C) – Wallace rule
o Anneal temp = Tm – 5 degrees celcius
Why does Tm increase with salt conc?
- Base stacking interactions
- Hydrophobic and electrostatic interactions between base pairs
- These interactions increase with increasing salt – the salt neutralizes the charge on the
phosphodiester backbone, thus allowing for stronger base-stacking interaction
PCR variant RPA: recombinase polymerase amplification
- binding of oligonucleotide is directed by a recombinase. Elongation uses a strand-
displacing polymerase (Bacillus subtilis Pol I (Bsu)).
- Recombinase is released from 3’ end upon hydrolysis of ATP. This allows the polymerase
to bind.
- Benefits
o Constant, low temperature (no special equipment needed)
o Speed: 5-10 minutes
o Specificity
o Rapid medical diagnostics, crop science, “field” use due to portability
How to kill RNAses
- Treat water with diethylpyrocarbonate (DEPC), reacts with histidine, hydrolyzes to CO2
and ethanol which is accelerated by amines – therefore cannot be used to treat Tris-
containing solutions.
- Heat glassware to 300oC for several hours
- Use RNAse inhibitors
- Set aside glassware, plasticware, pipets to be used only for RNA work.
- Tips and tubes should be certified RNAse-free.
Quantitative real time PCR
- fluorescence detection
- Fluorescence is monitored during the reaction and the number of cycles it takes to
reach a threshold value can be expressed as CT.
- CT is plotted against initial copy number for standards to yield a straight line from which
amounts can be directly inferred.
- This method is commonly used to compare two samples to detect the relative fold
increase or decrease of gene expression.
- PCR reaction phases: exponential, linear, plateau
