Nucleic Acids 3 Flashcards
What happens to dsDNA when it is melted and what condition is required for it to reverse?
Melting dsDNA above its melting temperature melts (denatures) it into two ssDNA.
Complementary sequences of ssDNA will hybridise (‘anneal’) spontaneously at temperatures of <65°C .
How is DNA replicated artifically?
dsDNA is melted at 95°C to split it into two ssDNA strands. They are then cooled at 50-65°C and a short DNA molecule (DNA primer) is annealed to each ssDNA. DNA polymerase is added and it copies the ssDNA strands by adding to the DNA primers to produce two dsDNAs. This can be repeated on the dsDNA produced to amplify exponentially. No. of molecules = 2^n where n is the number of rounds of this process.
How is DNA size measured and different sized DNA separated?
Using gel electrophoresis techniques. An electric field is applied across a hydrated polymer gel containing DNA fragments which causes the DNA fragments to migrate down the gel (to the side of the anode (as DNA is negatively charged)). Migration distance/speed/time depends on the DNA size due to the retarding effect of the gel.
What are potential advantages of using nanotechnology to sequence DNA?
High sensititvity (single molecule), longer read lengths, low error rate, reduced cost of personalised medicine.
How does gene transcription work?
RNA polymerase binds to the start sequence of the gene (the promoter). RNA polymerase generates a transcription bubble which turns into an RNA strand. RNA copy is released when the polymerase reaches the stop sequence of the gene.
How can G bases in DNA be detected?
STM can be used to detect G bases in DNA by measuring the conductance of a DNA molecule. Guanine bases have strong deviation of conductance from parabolic at -15V gap voltage. Guanine has a noticeable conductance (dI/dV) compared to the rest of DNA molecules.
How can different molecules be identified using STM?
Different molecules in STM tunnel gaps create different current/conductance vs distance/stretching relationships. These relationships can be used as fingerprints to identify specific molecules in the electron tunnel gap.
How can base pair sequences be identified with STM?
Pure G-C sequences show linear dependence on conductance with DNA length. Inserting an A-T base pair in the middle of the DNA sequence changes behaviour to exponential. This gives single base-pair resolution.
What are the limitations of using STM to investigate DNA?
As conductance and current decrease fast with increasing distance even without the presence of a molecule in the tunnel gap, only short DNA molecules can be investigated using STM.
STM tip can read along DNA bases and differentiate sequence but resolution is limited to about 20 bases because tip is far from surface.
Resolution of STM decreases with ________ distance between tip and sample.
Resolution of STM decreases with increasing distance between tip and sample.
How are synthetic nanopores made?
Using a focused ion beam (FIB) or focused electron beam (TEM).
How can DNA sequencing be perfomed using a nanopore?
By monitoring the ionic current passing through a nanopore with DNA passing through it. DNA is driven through the nanopore by an electric field (travels from region of negative charge to region of positive charge (which are on opposite sides of the nanopore)). Current through a nanopore is sequence dependent. Each of the 4 bases reduce the current passing through a nanpore by a different, specific amount. So current passing through a pore relative to current passing when pore is open can be used to identify the sequence of a DNA strand base-by-base.
DNA translocation through a nanopore is very fast. How can it be slowed down?
By applying a direct force with optical tweezers. By changing either electrolyte temperature, salt concentraion, solution viscosity, or the magnitude of the applied electric field.
Methylation of which DNA base is a marker of epigenetic traits on the genome?
Cytosine
What kind of bonds are used to make tunnelling recognition measurements of DNA in a STM tunnel junction?
Hydrogen bonds
