W1 - DNA & Hybridisation

Question 1

What is the molecular arrangement of DNA

Answer 1

• 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

Question 2

What are pyrimidines?

Answer 2

Cytosine and thymine (uracil)

Single ring structure

Question 3

What are purines?

Answer 3

Adenine and guanine

Double ring structure

Question 4

How does hydrogen bonding work with bases?

Answer 4

C - - - G
T - - A

Makes the bond dipole

Question 5

How are phosphates linked to the nucleotide?

Answer 5

They are linked with ester bonds creating phosphodiester bonds. 

Question 6

How are sugar phosphate link to the nucleotide?

Answer 6

Phosphodiester bonds

Question 7

What is base stacking?

Answer 7

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. 

Question 8

Why are the negatively charged phosphates outside?

Answer 8

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.

Question 9

What is denaturation?

Answer 9

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.

Question 10

How do you measure denaturation?

Answer 10

Optically -260nm absorbance
Hyperchromicity - increase absorption of light at 260 nm on denaturation.
At this point, 50% of all strands separate.

Question 11

What is melting temperature (Tm) ?

Answer 11

Depends on the composition of bases.
GC content, length of DNA molecule, salt concentration, pH (alkali = denaturant), mismatches 

Question 12

How does GC content affect melting temperature?

Answer 12

More GC = More H-bonds = higher Tm

GC% = (G+C)/(G+C+A+T) x100

Question 13

How does molecules length affect melting temperature?

Answer 13

Longer contiguous duplex = higher Tm
Higher H bonds = more stability

Little further contribution beyond 300bps 

Question 14

How does salt concentration affect melting temperature?

Answer 14

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.

Question 15

What effect does increasing salt have on specificity?

Answer 15

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.

Question 16

What affect does pH have on melting temperature?

Answer 16

Chemical denaturants distrupt hydrogen bonds (Alkali, formamide,urea)
OH- distrupt hydrogen bond pairing means fewer H bonds = Tm decreases.

High pH = destabilise DNA duplex

Question 17

How does mismatches affect temperature?

Answer 17

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.

Question 18

Why is duplex formation an equilibrium?

Answer 18

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.

Question 19

What is the difference between renaturation and hybridisation?

Answer 19

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.

Question 20

What is the relationship between complementarity and melting temperature & the basis of specificity?

Answer 20

Perfect matches = High Tm
This is thermodynamically favoured over mismatches.
Can use this property to form a complementarity molecule with no mismatches.

Question 21

What is stringency?

Answer 21

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.

Question 22

What does high and low stringency mean?

Answer 22

High = promotes formation of perfect matches
Low = promote formation of both perfectly matched and mismatched

Question 23

What are nucleic acid hybridisation techniques?

Answer 23

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.

Question 24

How does hybridisation use the ability of nucleic acids to form specific duplexes?

Answer 24

They use the complementarity and their preservation of labelled nucleic acid. These molecules are referred to as probes.

Question 25

What are probes?

Answer 25

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.

Question 26

What are some hybridisation based techniques?

Answer 26

• 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.

Question 27

What are genome wide techniques?

Answer 27

Hybridisation employed in: Microarrays + next-generation sequencing

Use for measuring gene expression/genomic composition

Question 28

What are microarrays?

Answer 28

Ordered assembly of thousands Nucleic acid probes. Probes of fixed solid surface example of interest is hybridised.



Question 29

What is next-generation sequencing?

Answer 29

Parallel sequencing of millions of molecules Captured in a surface by hybridisation.