DNA Hybridisation Flashcards
As a recap, describe the structure of a nucleotide.
The structure of a nucleotide includes a nitrogenous base, comprising of either a single or double ring containing nitrogen and carbon. The polar/charged groups on these are important for base pairing.
This ring structure is attached to a 5 carbon or pentose sugar (ribose) with at least one hydroxyl group attached to the 3’ position. Attached to the 5’ carbon of the ribose sugar is a phosphate group.
As a recap, describe the 4 nucleotides.
The 4 nucleotides fall into two molecular structures consisting of either a single or double nitrogen-containing ring (so either a pyrimidine (1) or a purine (2)).
An important difference in the structures resides in the charged or polar groups, providing the specificity of base pairing.
IN RNA, Uracil substitutes Thyamine and base pairs with Adenine in RNA to form a duplex structure.
What is Watson and Crick bonding?
Watson and Crick’s bonding is the pairing of nucleotide bases within DNA, and it is largely determined by hydrogen bonding between oppositely charged groups.
What is the consequence (bonding-wise) of the difference in structure in pyrimidines and purines?
An important difference between the pairs is the number of hydrogen bonds formed (GC has 3, while TA has 2).
As a consequence, the GC pairing is stronger than the TA pairing (and the UA pairing).
Give examples of three bondings in a DNA double helix that give it its stability.
- sugar phosphates: linked by phosphodiester bonds
- base stacking: hydrophobic interactions (the arrangement of bases as they are set above each other is internalised to the structure and excludes water)
- Van der Waals forces: individually small but they contribute to the stability
What are some ways in which DNA can get denatured?
- by the addition of chemicals (such as strong alkali or urea)
- by addition of heat
- basically, by disruption of the hydrogen bonds within the double helix
How can we measure denaturation?
Denaturation can be measured optically by absorbance at 260 nm.
Single-stranded DNA absorbs UV light to a greater extent than double-stranded DNA; this property is termed hyperchromicity.
The denaturation of a specific DNA depends upon the stability of the specific structure.
What is hyperchromicity?
Hyperchromicity is the increased absorption of light at 260 nm on denaturation.
What is the Tm?
The point at which 50% of all strands separate is called the melting temperature or Tm.
This characteristic is specific to an individual double helical structure, and we can use this knowledge to control the formation of the duplex.
What is the correlation between Tm and GC content?
The higher the GC content, the more hydrogen bonds, the higher the Tm.
%GC = (G+C)/(G+C+A+T) x 100
What is the correlation between Tm and molecule length?
The longer the continuous duplex, the more hydrogen bonds within the molecule giving it greater stability, the higher the Tm.
However, there is a diminishing return on this, and a length beyond about 300 base pairs contributes little or no more to the stability.
What is the correlation between Tm and salt concentration [Na+]?
Salt stabilises DNA duplexes.
Increasing the salt concentration stabilises the structure, increases the Tm, and overcomes the destabilising effect of mismatched base pairs, reducing the specificity of base pairing.
Explain the mechanism behind salt stabilising DNA.
DNA is a structure that is a balance between the repulsion of the charged backbones, and the attraction between the bases.
Adding salt reduces the charge repulsion, and shifts the balance towards attraction - increasing stability.
How does pH affect Tm?
Chemical denaturants can disrupt hydrogen bonds.
With fewer hydrogen bonds, the stability of the structure is lowered. Thus, a high pH destabilises DNA duplexes.
What is a mismatch?
A mismatch is defined as a base pair combination that is unable to form hydrogen bonds.
