ASSIGMENT

ON
HYPERCHROMICITY






















MEANING OF HYPERCHROMICITY -
Hyperchromicity is the increase of absorbance (optical density) of a
material. The most famous example is the hyperchromicity of DNA
that occurs when the DNA duplex is denatured. The UV absorption is
increased when the two single DNA strands are being separated,
either by heat or by of a chemical.
DNA's hyperchromic effect - that ss DNA absorbs more UV than
does ds DNA.
Example of hyperchromic effect - An insect, which can see in the UV
range, would see the hyperchromic effect something like that shown to
the below. At first the DNA solution is only a little violet. If it is boiled
and then slowly cooled, it ends up a little more violet than it started, but
if it is rapidly cooled it becomes most violet. The reason that this
happens is that in ds DNA the pi-electrons in the aromatic rings are more
o nstrained because the H-bonded rings are in sandwich layers -
overlapping with each other. But if the H-bonds are "boiled" away, the
sandwich no longer exists and the pi-electrons are more free to move
into different energy levels and thus able to absorb more UV energy.







Stability of the Double-Helix depend upon the following
interactions :
1) Hydrophobic Interactions: The ribose-phosphate is hydrophilic on
the outside of the chain. The bases are hydrophobic on the inside.
2) Stacking Interactions: Van der Waals forces Relatively weak but
additive Caused by the planar nature of the bases.
3) Hydrogen Bonding: Actually not the strongest of interactions.
Facilitates stacking.
4) Electrostatic Interactions: Both within chains and between chains.
Phosphate groups try to get as far away from each other as possible.
There are two basic approaches to denaturing double-stranded
DNA :
1) chemical treatment.
2) Heating
chemical denaturants can be divided into three classes :
a) pH: Bases like NaOH raise the pH until the H
+
shared between the N-
base electronegative centers ( N-H and O= ) is stripped from the H-
bond. Loss of H-bonds between two complementary strands results
in strand separation.
b) Competitive Denaturants : Compounds like urea and
formaldehyde contain functional groups that can form H-bonds with
the electronegative centers of the N-bases. At high concentrations
(8M urea or 70% formamide) of the denaturant, the competition for
H-bonds favors interactions between the denaturant and the N-bases
rather than between complementary bases. As a result, the two
strands separate.



c) Covalent Modification Denaturants : Reactive aldehydes like
formaldehyde and glyoxal can covalently modify the electronegative
centers of the N-bases and thereby block the formation of H-bonds
between complementary bases.Covalent modification is reversible.


1) Heat Denaturation :
Consider what happens when we heat a nucleic acid solution - say
the E. coli genome. To prepare the DNA for this experiment, we
shear it up into small pieces (approximately 500 bp long) and heat it
slowly while monitoring the A
260
.


As the temperature rises, the kinetic motion of molecules in solution
increases.The initial A
260
is stable until, over an interval of
approximately 5 degrees C, the A
260
suddenly increases by
approximately 40%.This increase in absorbance is referred to as the
Hyperchromic Shift.
REASON OF HYPERCHROMIC SHIFT :
The hyperchromic shift is due to the melting of the double helix into two single
strands. The increased rotational freedom of the N-bases on strand separation
accounts for the observed increase in absorbance.
DENATURATION OF DNA :
If we heat up a tube of DNA dissolved in water, the energy of the heat can pull
the two strands of DNA apart (there's a critical temperature called the Tm at
which this happens). This process is called 'denaturation'; when we've 'denatured'
the DNA, we have heated it to separate the strands.



Denaturation rate (i.e. the amount of denaturant required to do the DNA)
depends on adenine solubility -- not on the relative number of double and triple
hydrogen bonds.The more soluble adenine is in the denaturant, the less reagent
required to denature.AT-Rich region melt first because they have only two bonds.
Heat, pH, and temperature extremes will also destabilize and thereby denature
DNA.
DNA melting temperature ( Tm) :
For molecular biology applications such as PCR, sequencing or microarrays, it is
important to determine the melting temperature of DNA, or T
m
. The T
m
is defined
as the temperature in degress Celsius, at which 50% of all molecules of a given
DNA sequence are hybridized into a double strand, and 50% are present as single
strands.
Note that melting in this sense is not a change of aggregate state, but simply the
dissociation of the two molecules of the DNA double helix.
If a homogenous solution of identical double-stranded DNA molecules is heated,
the strands dissociate increasingly at higher temperatures:

In the example above, the Tm would be 60C.
The main factors which affect the melting temperature -
a) The GC content of the nucleic acid sample- This is due to the
fact that AT base pairs share 2 H-bonds while GC base pairs share 3
H-bonds.
b) SALT - Tm is sensitive to Na
+
concentration.Na
+
acts to shield the
negative charges of the sugar-phosophate backbone from
interacting with one another. The repulsion between the
negatively charged phosphate backbones is the major force
destabilizing the double helix, therefore increasing
Na
+
concentration increases helix stability and decreasing
Na
+
concentration decreases helix stability.
c) DNA hybrid length - The longer the DNA hybrid is, the more H-
bonds there are holding the two strands together. The longer the
hybrid, the more H-bonds that must be simultaneously broken for
the two strands to separate. This is known as the 'zipper effect'
after the (in) famous Canadian inventor Zippy. For our purposes
we will only consider the two extremes of the zipper effect. For
this course we will only consider the extemes of hybrid length -
hybrids less than 50 bp (short) and those around 500 bp (long) in
length.
D) The most complex factor is the sequence of the DNA. The
sequence has an impact on the Tm for a number of reasons:
The nucleotide pair A-T has a weaker bond than the nucleotide pair
G-C.
Nucleotides on the same strand can interact with each other, forming
so-called secondary structures such as internal loops; these
structures compete with the formation of the double helix and can
thus increase the Tm.
Neighbouring nucleotide pairs can interact with each other. It is energetically
favourable for nucleotide pairs to be neighboured with other nucleotide pairs. This
so-called stacking effect decreases the Tm.
Importance of hyperchromic effect -
The melting temperature (Tm), occurs almost instantly at a certain temperature,
monitoring the absorbance of the DNA at various temperature would indicate the
melting temperature. By being able to find the temperature at which DNA melted
and annealed, scientists are able to separate DNA strands and anneal them with
other DNA strands. This is important in creating hybrid DNAs, which consists of
two DNA strands from different sources. Since DNA strands can only anneal if
they are similar, the creation of hybrid DNAs can indicate similarities between
genomes of different organisms.
Tm is a measure of hybrid stability - The quantitative analyses of the melting
behaviour of many naturally occuring DNAs is summarized in the formula (note:
this formula describes the Tm of long DNA hybrids)
Tm = 81.5
o
C + 0.41 ( % GC ) + 16.6 log [Na
+
]
note that the % GC term only holds over a limited range (45 to 75%) as does the
salt term ( 0.01 to 0.4 M ). note that the salt term is negative in this range.
For short DNA hybrids, the 'zipper effect' alters the formula to
Tm = 2
o
C (A+T) + 4
o
C (G + C)
Renaturation of DNA - The reannealing of two single strands is a bi-
molecular reaction. Two complementary single strands must meet one another
and then complementary base pairs form between the two strands. Because this is a
bi-molecular reaction, the rate of the reaction depends on the concentration of the
reactants - the two complementary strands.




.





Renaturation of DNA




The following example for cot analysis -
Reannealing reaction conditions are therefore chosen to maximize the rate at
which hybrids form. The curve below shows how the rate of reannealing depends
on temperature. To maximize the rate of reannealing, we typically hybridize 15
o
C
below the Tm of the hybrid. Hybridization is also done at high salt to minimize the
repulsion of the sugar-phosphate backbones.
Hybridization conditions are always set to maximize the rate of hybrid formation.
The rate at which hybridization occurs is therefore a direct measurement of the
concentration of the complementary strands in solution. In order to visualize this
concept, consider the reannealing of a variety of DNA samples.
To standardize conditions :
Each DNA sample is sheared to random fragments 500 bp long.
Each DNA sample is at the same DNA (50 ug/ml) and NaCl concentration(1M).
Each sample is heated to boiling to denature it and then held at (Tm-15
o
C) while
the amount of DNA remaining single stranded is monitored.




The % single stranded is graphed as a function of
Cot - the initial DNA concentration (Co) times time (t)
Since the reaction follows bimolecular kinetics, we can obtain a value
Cot
1/2
= Cot at which 1/2 of the DNA has reannealed
Cot
1/2
measures the rate of reannealing which is inversely proportional to the
concentration of complementary sequences being examined.
Small Cot
1/2
values indicate that the complementary sequences are at high
concentration.
(they reanneal very quickly, t is small)
Large Cot
1/2
values indicate that the complementary sequences are at low
concentration.
(they reanneal very slowly, it is large)
The lambda genome consists of 50,000 bp of unique sequence.The DNA
reanneals as a single kinetic component (a single sigmoidal curve). The midpoint
of the curve provides the value for Cot
1/2
- the time required for half of the single
strands to be in double stranded form at a defined initial DNA concentration.

In contrast to the above example, the E. coli genome consists of 5,000,000 bp of
unique sequence. Again, plotting the % single stranded as a function of Cot, we
observe a single sigmoidal curve confirming that the E coli genome reanneals as a
single kinetic class.

Notice that the Cot curve has shifted to the right relative to the lambda Cot curve.
The increase in Cot
1/2
reflects the number of copies of the E coli genome in
solution as compared to the lambda genome. Since the E coli genome is 2 orders
of magnitude larger than the lambda genome, any given 500 bp fragment will be
present at 100 x lower concentration in the E coli genome sample.
This can be illustrated by considering what happens when the DNA sample
consists of equal mass amounts of the lambda and E coli genomes (each at 25
ug/ml)

The Cot curve now has two kinetic components,each comprising about 1/2 of
the total DNA in the sample :
a) one with a Cot
1/2
of the lambda genome,
b) one with a Cot
1/2
of the E coli genome.
IN CASE OF EUKARYOTES :
Unlike most prokaryotes, the Cot curves of eukaryotic genomes are complex
curves. Modeling based on bimolecular kinetics allows us to 'fit' the data to define
three kinetic classes that differ in their repetition frequency in the genome.
a) The first class represents a small portion of the genome (typically 10% or
so) but is very highly repeated (10,000s of copies). These are short highly
clustered repeated sequences found at eukaryotic telomeres and
centromeres. Dispersed copies of these simple sequence repeats are also
common.

b) The second class is called moderately repetitive. This class contains longer
sequences which are repeated 100s to 1000s of times in the genome.
Ribosomal RNA genes, histone genes and a few others fit into this class.
Another major component are the transposable elements - a class of
sequence which has the capacity to replicate and move to new positions in
the genome. The portion of the genome in this portion of the Cot curve
varies widely.
c) The third kinetic class is the unique sequence of the genome. In this class
we find most the protein coding sequences of the genome. This class
typically contains the majority of the sequence in the eukaryotic genome.