Denaturation and Renaturation of DNA

Hydrogen bonds in double-stranded DNA (double strand DNA, dsDNA) can be broken by
extreme pH, urea or heat. The subsequent separation of the chains, i.e. DNA denaturation,
can be monitored using the so-called hyperchromic effect. Due to the interaction of electrons
in paired bases, dsDNA solutions absorb UV light (at 260 nm) less than the same concentration
of bases in mononucleotides or in single-stranded DNA (single strand DNA, ssDNA). When
the dsDNA solution is heated, the absorbance suddenly rises at a certain temperature. The
temperature at which the increase in absorbance reaches its half is called the melting point of
DNA (melting point, Tm). The more G=C pairs the examined dsDNA contains, the higher the
Tm. Three hydrogen bonds in G=C require more thermal energy than two in A=T.

DNA denaturation is reversible. Complementary ssDNA can rejoin into dsDNA when, for
example, the temperature drops or the concentration of urea decreases. Unfolding and folding
of the double helix is a common phenomenon in vivo (e.g. in the biosynthesis of nucleic acids).
Fusing of two complementary strands of nucleic acids in vitro into double helices is
called reassociation. If different types of nucleic acids reassociate (DNA with RNA), it is
called molecular hybridization. Methodically, these phenomena are used to separate a certain
ssDNA or ssRNA from a mixture, to map the genome and to detect disorders of
the gene structure (diagnosis of genetic defects).

Denaturation and Renaturation is the characteristic of DNA molecules. The factors like
temperature, pH and chemical agents form or break the hydrogen bonds present between the
two strands of DNA. It is a reversible process and it is used to study the complexity of the
genome. Genetic Research & such studies use DNA Denaturation and Renaturation processes.
Denaturation is defined as change in the native structure of the biomolecule. Breaking of non
covalent bonds like hydrogen, ionic and van der waals forces etc causes the denaturation. The
DNA is right handed helical structure, it consist of two polynucleotide that are hold together
by hydrogen bonds that are formed between the complementary nitrogen base pairs. DNA
denaturation means the breaking of hydrogen bonds that causes separation of two strands.
The hydrogen bond is the force of attraction between hydrogen atom of one covalently
electronegative atom or group with other electronegative atom or group. It is weak bond and
its strength ranges from 4 kJ to 50 kJ per mole. The strength of hydrogen bond is only 5% as
compared to covalent bond. Due to the weakness, hydrogen bonds are prone to break. Different
factors like temperature, pH and chemical agents can cause the breaking of hydrogen bonds.
And hence, denaturation is also affected by the same factors.
Electropositive and Electronegative.

Source: Lehninger, Nelson and Cox

Temperature:
The temperature more than 80°C causes DNA denaturation by breaking the hydrogen bonds
and producing two separate strands. The temperature at which DNA denature is called as
melting point of DNA or melting temperature (Tm). The melting point is characteristic of DNA
because Tm is affected by ATGC content. The sequence with more AT content would have less
Tm then the DNA sequence with GC. It is because GC has triple bonds and hence more
temperature or energy is required to break three bonds than two.
pH:
The increase or decrease pH causes changes in ionic charges of nitrogen bases (purine and
pyrimidines). At low pH, the amino groups become protonated and high pH causes its
deprotonation. Both the conditions are not favorable to form the hydrogen bonds and hence it
disrupts the native structure of DNA and lead to denaturation.

Chemical agents:
Urea and formamide or formaldehyde is known to break the hydrogen bonds of DNA helix.
These chemicals tend to form hydrogen bond with the electronegative centers of nitrogen bases.
This causes disruption of the native hydrogen bonds between complementary base pairs of
DNA structure. The melting point of DNA or Tm can be altered by adding such chemicals.

Characteristics of Denatured DNA

1) Viscosity: The double stranded helical structure is viscous in natured. When DNA is
denatured by any of the mentioned factor, it loosed it viscosity.
2) Optical rotation: The native structure of DNA is optically activity. The denaturation of DNA
causes decrease in the optical activity.
4) UV absorption: The native structure of DNA absorbs UV light at 260nm because of the
aromatic rings of nitrogen bases. When DNA denatures the absorbance of UV light increase
because of the exposure of Nitrogen bases and it is called as hyperchromic shift or
hychromacity.
Hence the extent of denaturation of DNA can be observed of study by the UV light absorption.

Renaturation:
When the two separated strands of DNA bind together again, it is called as renaturation of
DNA. It means it is transformation from denatured structure to native one. Renaturation of
DNA occurs at room temperature or neutral pH or in the absence of denaturating chemical
agents. The low temperature reduces the entropy and favors the colliding and contact of the
two strands. When two strands come in to close proximity, they tend to form hydrogen bonds
again forming double stranded helix. Hence Denaturation is reversible process.
When denatured DNA is renatured, the absorbance of UV at 260 nm decreases because of
concealing of nitrogen bases. And this is called as Hypochromic shift.
The Single stranded DNA also tends to form hydrogen bonds with SS DNA from different
source. It happens when SS DNA from different source have complementary sequence. This
process is called as hybridization. It is used to study the evolutionary relationship of different
species.
The renaturation of depends on concentration of DNA and time. Higher the concentration of
DNA, faster is the renaturation. And hence the term is given as Cot (concentration x time)
The plot depicts the renaturation of double stranded DNA of prokaryotic cell.

Source: Lehninger, Nelson and Cox

The plot depicts the renaturation of double stranded DNA of prokaryotic cell. The cot
(concentration of DNA x time) is on X-axis and Percentage or fraction of SS DNA is on Y-axis.
The curve that starts from upper left hand of Y-axis indicates the starting of renaturation
reaction. 1 or 100% on X-axis means that the entire DNA is present in single stranded form.
As the time passes, the DNA strands reanneal and the proportion or percentage of single
stranded DNA decreases. When the entire DNA gets reannealed or renatured, the proportion of
SS DNA become zero. The point at which half of the DNA is renatured, it is called as Cot1/2.
DNA contains unique and repetitive sequences. Unique means genes for particular character,
which are present only once, or twice in DNA. Repetitive sequence means a stretch of
nucleotide that is repeated multiple times in DNA. Britten and Davidson have found that higher
the number of repetitive sequence present in DNA faster is the renaturation or annealing
process. As the same sequence is in multiple copies, the probability of colliding with its
complementary sequence becomes more. The repetitive sequences could be highly or
moderately repeated.
To understand above statement, lets take an example. Imagine a tub with red, blue and white
balls. Suppose, the box has 60 Red balls, 30 blue balls and 10 white balls. If you randomly try
to pick these balls the probability of picking red balls is more than blue balls and much more
than white balls. In the similar way, the collision of repetitive sequence with each other is more
than unique sequence.
Hence, based on types of sequence (highly repetitive, moderately repetitive or unique genes)
present in DNA affect the rate of renaturation. Rate of renaturation is directly proportional to
number of repetitive sequences. The Cot analysis is used to measure repetitive sequences and
complexity of genome.