• The nitrogenous bases are paired in specific

combinations: adenine with thymine and

guanine with cytosine.
• Pairing like nucleotides did not fit the uniform
diameter indicated by the X-ray data.
– A purine-purine pair would be too wide and a
pyrimidine-pyrimidine pairing would be too short.
– Only a pyrimidine-
purine pairing would
produce the 2-nm
diameter indicated
by the X-ray data.

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Chargaff's rules

One other key piece of information related to the structure of DNA came
from Austrian biochemist Erwin Chargaff. Chargaff analyzed the DNA of
different species, determining its composition of A, T, C, and G bases. He
made several key observations:

A, T, C, and G were not found in equal quantities (as some models at the
time would have predicted)

The amounts of the bases varied among species, but not between
individuals of the same species

The amount of A always equalled the amount of T, and the amount of C

always equalled the amount of G (A = T and G = C)

These findings, called Chargaff's rules, turned out to be crucial to Watson

and Crick's model of the DNA double helix.
Denaturation X Renaturation
of DNA
When a DNA solution is heated enough, The double-
stranded DNA unwinds, and the Hydrogen bonds that
hold the two strands together weaken and finally
break. The process of breaking a double-stranded
DNA into single strands is known as DNA
denaturation, or DNA melting.
The temperature at which the DNA strands are
half denatured, meaning half double-stranded,
half single-stranded, is called the melting
temperature(Tm)

The amount of strand separation, or melting, is

measured by the absorbance of the DNA
solution at 260nm. 
 What determines the Melting Temperature (Tm)?
While the ratio of G to C and A to T in an organism's DNA is fixed, the GC
content (percentage of G +C) can vary considerably from one DNA to
another.

The percentage of GC content of DNA has a significant effect on its Tm.

Because G-C pairs form three hydrogen bonds, while A-T pairs form only
two, the higher the percentage of GC content, the higher its Tm.

Thus, A double-stranded DNA rich in G and C needs more energy to be

broken than one that is rich in A and T, meaning higher melting
temperature(Tm).

Above the Tm, DNA denatures, and below it, DNA anneals. Annealing is the
reverse of denaturation.
The Temperature of Melting (Tm) is defined as
the temperature at which 50% of double stranded
DNA is changed to single-standard DNA.

The higher the melting temperature the greater

the guanine-cytosine (GC) content of the DNA.
Heating is not the only way to denature DNA.

Organic solvents such as dimethyl sulfoxide and

formamide, or high pH, could break the hydrogen
bonding between DNA strands too.

Low salt concentration could also denature DNA

double-strands by removing ions that stabilize the
negative charges on the two strands from each other.