Sept 22, 2009
Single Stranded DNA Genomes
-Some organisms use a single strand of DNA as their form of genetic information
storage; only during replication is the genome in a double stranded form
-There is an intermediate form of the genome called the replication form (RF)
-The standard double stranded nature of most genomes can be converted into a
single stranded form by heat denaturation
Native ds DNA –(100°C `5min) denatured state of ss DNA
-The amount of DNA that is denatured can be measured via a spectrometer using
a 260nm wavelength light. The absorbency of the light is dependent on the
structure of the strands themselves, the more bases that are exposed will result in
a grater amount of absorbency. The amount of base stacking that occurs when the
double helix is broken is greatly reduced, this increases the absorbency of DNA.
-Refer to previous notes for DNA melting
Evidence That H-Bonds are Important
-Plot melting temperature (Tm) vs. GC content; Tm will increase as the amount of
CG content increases (CG pairs have 3 H-bonds vs. AT pairs that only have 2)
-Tm in the presence of foramide or urea causes the Tm to decrease because of the
competition that occurs between these chemical agents and the H-bonding
between the nucleotides.
-An increase in pH causes the Tm to decrease because (when pH > 11.3 DNA is
fully denatured) the structure of the molecule becomes unstable as the pH rises.
The pKa values of DNA comes into play once the pH approaches or exceeds that
value since deprotonation occurs and H-bonding becomes unviable.
Evidence That Base Stacking is Important
-As Na-trifluoroacetate concentration increases the Tm decreases, this is due to
the fact that it disrupts the water shell around a hydrophobic substance; this leads
to less hydrophobic interactions.
-Alcohols and amines decrease the Tm because the solubility of the bases
increases which also lowers the hydrophobic interactions
Forces That Destabilize the Structure of ds DNA -PO 4roups destabilize the double helix structure due to the negative charge of
the phosphates, however, increasing the amount of NaCl will increase