W1 - DNA & Hybridisation Flashcards
What is the molecular arrangement of DNA
- pentose sugar – five carbons that form a cylindrical structure with oxygen bridge.
- nitrogenous base - ring structure composed of carbon and nitrogen join to carbon 1.
- phosphate group joined to carbon 5
- hydroxyl group and carbon 3
What are pyrimidines?
Cytosine and thymine (uracil)
Single ring structure
What are purines?
Adenine and guanine
Double ring structure
How does hydrogen bonding work with bases?
C - - - G
T - - A
Makes the bond dipole
How are phosphates linked to the nucleotide?
They are linked with ester bonds creating phosphodiester bonds. 
How are sugar phosphate link to the nucleotide?
Phosphodiester bonds
What is base stacking?
Hydrophobic interactions create this arrangement.
interactions ->arrangement of bases set above each other internalised to the structure & excludes water
This further stabilises the structure.
Van der Waals forces are individually small but contributes to the stability. 
Why are the negatively charged phosphates outside?
Antiparallel strands from 5’ to 3’ prime orientation.
The phosphate backbone of DNA is negatively charged, which is due to the presence of bonds created between the phosphorus and oxygen atoms. In DNA structure, a phosphate group comprises one negatively charged oxygen atom, which is responsible for the entire strand of DNA to be negatively charged.
What is denaturation?
Destruction of hydrogen bonds within double helix.
Occurs when DNA in solution is heated or can be induced by a strong alkali or urea.
Double helix once denatured forms random coil.
How do you measure denaturation?
Optically -260nm absorbance
Hyperchromicity - increase absorption of light at 260 nm on denaturation.
At this point, 50% of all strands separate.
What is melting temperature (Tm) ?
Depends on the composition of bases.
GC content, length of DNA molecule, salt concentration, pH (alkali = denaturant), mismatches 
How does GC content affect melting temperature?
More GC = More H-bonds = higher Tm
GC% = (G+C)/(G+C+A+T) x100
How does molecules length affect melting temperature?
Longer contiguous duplex = higher Tm
Higher H bonds = more stability
Little further contribution beyond 300bps 
How does salt concentration affect melting temperature?
Salt stabilises DNA duplexes.
High [Na+] = Increase Tm
Increase in salt concentration stabilises structure, increasing melting temperature and overcomes destabilising effect of mismatched base pairs.
What effect does increasing salt have on specificity?
Increasing salt reduces specificity of base pairing at a given temperature.
With low salt the duplex is unstable.
With high salt the same duplexes stable at the same temperature.
What affect does pH have on melting temperature?
Chemical denaturants distrupt hydrogen bonds (Alkali, formamide,urea)
OH- distrupt hydrogen bond pairing means fewer H bonds = Tm decreases.
High pH = destabilise DNA duplex
How does mismatches affect temperature?
Mismatching means base pair combinations Are unable to form H bonds. Decrease the number of hydrogen bonds means decreasing melting temperature. Shorter continuous stretches of double-stranded sequences means decreasing Tm.
Mismatches distort structure and destabilises adjacent base pairs.
Why is duplex formation an equilibrium?
Can have movement in both directions.
Denaturation and renaturation can be promoted depending on manipulation.
Formation of structure favours energy minimisation driven by changing free energy ^G . Facilitated by slow cooling and neutralisation.
What is the difference between renaturation and hybridisation?
Duplex denaturation means separation of strands. Renaturation is those strands being reconnected.
Hybridisation is when the old strand gets combined with a different organism’s DNA.
What is the relationship between complementarity and melting temperature & the basis of specificity?
Perfect matches = High Tm
This is thermodynamically favoured over mismatches.
Can use this property to form a complementarity molecule with no mismatches.
What is stringency?
Manipulating conditions- limiting hybridisation between imperfectly matched sequences allow us to manipulate specificity. 
Increasing stringency:
Only complimentary sequences are stable determined by:
Temp near Tm and salt conc being low.
What does high and low stringency mean?
High = promotes formation of perfect matches
Low = promote formation of both perfectly matched and mismatched
What are nucleic acid hybridisation techniques?
Identifies the presence of nucleic acids containing a specific sequence of bases.
This allows the absolute or relative quantitation of these sequences in a mixture.
How does hybridisation use the ability of nucleic acids to form specific duplexes?
They use the complementarity and their preservation of labelled nucleic acid. These molecules are referred to as probes.
What are probes?
SSDNA or RNA molecule.
Typically 20 to 1000 bases in length.
Labelled with a fluorescent/luminescent molecule (less commonly radioactive isotope).
It’s some techniques thousand/millions of probes are used simultaneously.
What are some hybridisation based techniques?
- in situ hybridisation of Tissue sections
- Chromosome painting of chromosome spread
-Analysis of mRNA / DNA by PCR / qPCR - Sanger/dideoxy sequencing
These techniques are not very scalable - can only detect few genes at once.
What are genome wide techniques?
Hybridisation employed in: Microarrays + next-generation sequencing
Use for measuring gene expression/genomic composition
What are microarrays?
Ordered assembly of thousands Nucleic acid probes. Probes of fixed solid surface example of interest is hybridised.

What is next-generation sequencing?
Parallel sequencing of millions of molecules Captured in a surface by hybridisation. 
