DNA Denaturation and Renaturation, Cot curves

1. DNA Denaturation and
Renaturation, Cot curves

2. Absorption of UV light
Nucleic acids exhibit characteristic absorption in the ultraviolet region. This
absorption is due to the conjugated double bonds and ring system of constituent
purine and pyrimidines.
The more ordered the structure, the less light is absorbed.
Therefore, free nucleotides absorb more light than a single-stranded polymer of DNA
or RNA and these in turn absorb more light than a double-stranded DNA molecule.
The maximum absorption is at 260 nm (A260) and the minimum absorption is at 230
nm.
Absorption is proportional to the concentration of the molecule, with a value of 0.02
units per μg DNA per ml.
For example, three solutions of double-stranded DNA, single-stranded DNA and free
bases each at 50 μg/ml have the following A260 values:
• Double-stranded DNA A260 = 1.00
• Single- stranded DNA A260 = 1.37
• Free bases A260 = 1.60
Therefore, double stranded DNA is said to hypochromic and the bases are said to be
hyperchromic.

3. Denaturation of DNA Molecules
The ordered state of DNA helix, which is, originally present in nature is called the native
form.
The two strands of DNA readily come apart when the hydrogen bonds between its paired
bases are disrupted. This can be accomplished by heating a solution of DNA or by adding
acid or alkali to ionize its bases. This unwinding of DNA double helix is called melting and
a transition from the native to the denatured state is called denaturation.
Denaturation of DNA molecule can be studied by measuring its absorbance at a
wavelength of 260 nm.
As the DNA is subjected to an increase in temperature, A260 starts increasing because of
DNA.
When both the strands are completely separated at a particular higher temperature,
there is maximum A260 that indicates complete denaturation of the molecule has taken
place.
The temperature at which half of the helical structure of DNA molecule is lost is called its
melting temperature (Tm). A convenient parameter to analyze melting transition.
Molecules rich in GC pairs have a higher Tm than those having abundance of AT base
pairs because GC base pairs are more stable and held together by three hydrogen bonds.
Such DNA molecules require more energy and hence temperature to denature.

4. Denaturation involves changes
Denaturation converts the firm, helical two-stranded native structure of DNA to a flexible,
single-stranded denatured state.
The splitting of DNA molecule into its two strands or chains is obvious because of the fact
that the hydrogen bonds are weaker than the bonds holding the bases to the sugar
phosphate groups.
Denaturation involves following changes :
Increase in absorption of ultraviolet light: Due to resonance, all of the bases in nucleic
acids absorb ultraviolet light. And all nucleic acids are characterized by a maximum
absorption of UV light at wavelength near 260nm. When the native DNA (which has base
pairs similar to a stack of coins) is denatured, there occurs a marked increase in optical
absorbance of UV light by pyrimidine and purine bases, an effect called hyperchromicity or
hyperchromism which is due to unstacking of the base pairs. This change reflects a
decrease in hydrogen bonding.
Decrease in specific optical rotation: Native DNA exhibits a strong positive rotation which is
highly decreased upon denaturation. (same as in proteins)
Decrease in viscosity: The solutions of native DNA possess a high viscosity because of the
relatively rigid double helical structure and long, rodlike character of DNA. Disruption of
the hydrogen bonds causes a marked decrease in viscosity

5. For example (the absorption of ultraviolet light),
if a solution of double-stranded DNA has a value
of A260=1.00, a solution of single-stranded DNA
at the same concentration has a value of
A260=1.37.
This relation is often described by stating that a
solution of double-stranded DNA becomes
hyperchromic when heated.
The following features of this curve should be
noted:
 The A260 remains constant up to temperatures well above those
encountered by most living cells in nature.
 The rise in A260 occurs over a relatively narrow range of 6-8℃
 The maximum A260 is about 37% higher than the starting value
Please note that during melting all covalent bonds, including
phosphodiester bonds, remain intact. Only hydrogen bonds and
stacking interactions are disrupted.
Denaturation and absorbance

6. Compounds like urea and formamide/formaldehyde are capable of hydrogen bonding
with the DNA bases. Hence, they maintain the unpaired state of DNA molecules and result
in lowered Tm value, upon melting.
Formaldehyde reacts with NH2 groups DNA bases and eliminates their ability to hydrogen
bond. Hence addition of formaldehyde causes a slow and irreversible denaturation of
DNA.
There is always a fluctuation in the structure of DNA. The double-stranded regions
frequently open to become single-stranded bubbles. This phenomenon is called breathing,
which enables specialized proteins to interact with DNA molecule and to read its encoded
information.
Breathing occurs more often in regions rich in AT pairs than in regions rich in GC pairs.
There are many proteins that can unwind a DNA helix. An example of this type of protein
is gene 32 of E. coli phage T4, commonly called the 32-protein. This protein binds tightly to
the bases of single-stranded DNA. The individual molecules of the 32-protein prefer to line
up adjacent to one another along a single strand. Binding of the first molecule is made
possible by the breathing of the DNA.
Denaturation of DNA can also be accomplished by treatment with alkali. Since DNA is
quite resistant to alkali hydrolysis, this procedure is the method of choice for denaturing
DNA, because heat treatment may often break the phosphodiester bonds and may result
in yielding broken fragments of DNA.
How can we achieve Denaturation

7. DNA Renaturation
• Denatured DNA will renature to re-form the duplex structure if the denaturing
conditions are removed (that is, if the solution is cooled, the pH is returned to
neutrality, or the denaturants are diluted out).
• Renaturation requires re-association of the DNA strands into a double helix, a
process termed reannealing.
• For this to occur:
(1) Strands must realign themselves so that their complementary bases are once
again in register (NUCLEATION PROCESS)
(2) Helix can be zippered up (Figure 12.19).
• Renaturation is dependent on DNA concentration and time.
Many of the realignments are imperfect, and thus the strands
must dissociate again to allow for proper pairings to be
formed.
• The process occurs more quickly if the temperature is warm
enough to promote diffusion of the large DNA molecules but
not so warm as to cause melting.

8. Renaturation Rate and DNA
Sequence Complexity—C0t Curves
• The renaturation rate of DNA is an excellent indicator of the sequence
complexity and the size of the DNA.
• For example, bacteriophage T4 DNA contains about 2x105 nucleotide
pairs, whereas Escherichia coli DNA possesses 4.64x106. E. coli DNA is
considerably more complex in that it encodes more information. Or we
may say that for any given amount of DNA (in grams), the sequences
represented in an E. coli sample are more heterogeneous, that is, more
dissimilar from one another, than those in an equal weight of phage T4
DNA. Therefore, it will take the E. coli DNA strands longer to find their
complementary partners and reanneal. This situation can be analyzed
quantitatively.

9. • If c is the concentration of single-stranded DNA at time t, then the second-order rate
equation for two complementary strands coming together is given by the rate of
decrease in c:
-- dc/dt = k2c2
where k2 is the second-order rate constant.
• Starting with a concentration, C0, of completely denatured DNA at t 0, the amount
of single-stranded DNA remaining at some time t is
C/C0 = 1/(1 + k2C0t)
where the units of C are moles of ntd per L and t is in seconds.
• Then the time for half of the DNA to renature (when C/C0 = 0.5), according to the
second order rate equation, is defined as t = t1/2. Then,
0.5 = 1/(1 + k2C0t 1/2) and thus 1 + k2C0t 1/2 comes out to be 2
yielding C0t 1/2 = 1/k2
• A graph of the fraction of single-stranded DNA reannealed (C/C0) as a function of C0t
on a semilogarithmic plot is referred to as a C0t (pronounced “cot”) curve (Figure).
DNA Sequence Complexity and C0t Curves

10. C0t Curves