Analysis of Nucleic acids

Question 1

1. Absorption of UV light

Answer 1

1. Nucleic acids absorb UV light within the range 254–260nm
   as a result of the aromatic rings of the purine and pyrimidine
   bases in nucleic acid.
2. Peak absorption is at 260 nm in an aqueous system.
   As a result, we can use UV light in the:
   > localization
   > Isolation
   > and characterization of nucleic acids.
   It can also be used to determine the amount of nucleic acids in solution.
   BECAUSE:
   At an absorption value (optical value) of 1 = [DNA] of
   50 ng/microL

Question 2

2. Other analyses of nucleic acids are based off of:

Answer 2

The hydrogen bonding between bases
> Denaturation:
(breaking H-bonds) induced by heat, pH changes or chemical treatments
- any treatment that causes destabilization between the base pairs

> Renatured
(complementary strands rejoin)

Question 3

Hyperchromic shift:

Answer 3

Increase in UV absorption and buoyancy of DNA
Due to:
Denaturing- when secondary structure is lost ( double helix)

Opposite: Hypochromatism
(when is double helix form) (-) UV abs.

Question 4

Tm (Melting temperature)

Answer 4

Temp at which half of DNA is in double Helix form and other half is in a random coiled state

Temp at the midpoint of the melting curve

Question 5

Used to determine Melting profiles of DNA

Answer 5

Tm

• because of H bonding, this is also dependent on the GC content of the DNA
• Because G-C has 3 H bonds, the higher the content of G-C base pairs in DNA = higher melting point of DNA

Thus, higher Tm = high G-C content

Question 6

Molecular hybridisation

Answer 6

• When conditions that cause denaturation are removed, random collisions of complementary strands will result in renaturation.
  > (re-association) of complementary single strands of nucleic
  acids.
  > H bonds stabilise = double-stranded structure
• The strands don’t have to come from original DNA molecule, there are enough complementary regions to form stable DNA molecules

DNA from different organisms can also be mixed - if enough base complementarity = Hybrid double-stranded DNA molecule.

This can happen between DNA and RNA molecules
= Molecular hybridization
> allows us to ID complementary DNA sequences and to locate specific genetic info

One of the most powerful analytic techniques in molecular biology. Applications include FISH, PCR, and Southern Blotting.

Question 7

Gel electrophoresis: