Nucleic Acids Flashcards
What is Chargaff’s Rule?
That in DNA the amount of dATP and dTTP is equal, and the amount of dGTP and dCTP is equal, and thus A-T and G-C must base pair.
What is the mechanism of ligation by DNA ligase? What conditions is this carried out in and why?
1) The ligase is adenylated, by the adding of AMP (from ATP or NAdD+) to a lysine residue of the ligase.
2) This AMP is transferred to the 5’ phosphate, activating it.
3) AMP is displaced which seals the nick in the phosphodiester backbone.
This is carried out at a sub-optimal temperature, to slow down the molecular movements of DNA to ensure that the ligase has enough time to find the nick and transfer the AMP.
What does alkaline phosphatase do?
Removes the 5’ phosphate from DNA or RNA, meaning that the ends become resistant to ligation. The 5’ phosphate is replaced with an -OH group. This is useful in DNA cloning, but is not 100% efficient.
What is hyperchromicity?
The increase in absorbance of a material. For example, the absorbance at 260nm increases when dsDNA goes from an ‘ordered’ to a single stranded ‘disordered’ structure, as the bases become unstacked and thus can absorb more light.
What does polynucleotide kinase do?
It transfers the terminal (gamma) phosphate of ATP to the OH of the 5’-terminus of DNA/RNA (that was created by the alkaline phosphatase). This is useful if you want to create radiolabelled DNA.
What is ethidium bromide used for? Why would you use an alternative?
It is used to make the DNA visible in gel electrophoresis, by intercalating between stacked bases and fluorescing when exposed to UV light. May use an alternative as it is a potential carcinogen, so can use Gel Red which is too big to cross the skin barrier.
Explain the Southern blot process.
1) DNA is cleaved and electrophoresis used to separate DNA
2) DNA fragments are blotted onto nitrocellulose filter
3) Filter is put in a heat-sealed bag and exposed to the radioactive probe (in solution). The probe will hybridise with homologous DNA fragments
4) The filter is exposed to photographic film and the film is developed, and bands on the x-ray film will have been created by the binding of the labelled probe
What is a DNA library?
A library is a collection of bacterial colonies that each contains a different fragment of the target DNA.
Describe what the function of a topioisomerase is.
To relax supercoiled DNA by introducing transient single-stranded or double-stranded breaks into DNA. This may be to undo excessive winding that may be preventing access to enzymes for instance. They can also increase supercoiling (rewind the DNA).
