Factor affecting structure of DNA, denaturation.

2.  DNA is a double helix structure. It is a molecule that carries
the genetic instructions used in the growth, development,
functioning and reproduction of all known living organisms and
many viruses.
 The two DNA strands are termed polynucleotide's since they are
composed of simpler monomer units called nucleotides.
 Each nucleotide is composed of one of four nitrogen-containing
nucleobases—either cytosine , guanine, adenine, or thymine —
and a sugar called deoxyribose and a phosphate group.
 The nucleotides are joined to one another in a chain by covalent
bonds between the sugar of one nucleotide and the phosphate,
resulting in an alternating sugar-phosphate backbone.
 The two strands of DNA run in opposite directions to each other
and are thus antiparallel.
 The DNA double helix is stabilized primarily by two
forces: hydrogen bonds between nucleotides and base-
stacking interactions among nucleobases.

4. There are various factors that affect the
structure of DNA:
 Elevated temperature
 Extremes of Ph
 Nonphysiological concentrations of salt,
organic solvent, urea or any other chemical
agents.

5.  DNA denaturation is the seperation of double
strand into two single strands, which occurs
when the hydrogen bonds between the
strands are broken.

6.  The most common type of denaturation is thermal
denaturnation.
 The temperature at which the DNA strands are half denatured,
meaning half double-stranded, half single-stranded, is called
the melting temperature(Tm).
 Below Tm the DNA is not denatured it remains double
stranded. Therefore, Tm is also known as the critical
temperature.
 Tm depends on:
• base composition
• length
• ionic strength
• pH
• denaturing agents

7. Further increase in temperature causes
steep rise in the absorbance followed by
another plateau as the DNA gets
completely denatured at these
temperatures

8.  At high pH the hydroxide ions (negatively
charged ions) can pull hydrogen ions from base
pairs-forming H bond between two strands-
causing them to separate.
 High pH will alter the charged groups that are
involved in H-bond.
 H-bond cannot occur at
pH>11.3 and DNA
denatures.

9.  Low salt concentrations could also denature DNA double
strands by removing ions that stabilize the negative charges
on two strands from each other.
 Compounds like urea and formaldehyde contain functional
groups that can form H-bonds with the electronegative
centers of the N-bases. At high concentrations 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.
 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.

10. These chemical reagents enhance the
aqueous solubility of the purine and
pyrimidine groups. The Tm value is lowered
by the addition of urea. In 8M urea, Tm is
decreased by nearly 20°C. DNA can be
completely denatured by 95% formamide at
room temperature only.

11.  Increase in Absorption of UV-Light:If denaturation is
followed spectrophotometrically by monitoring the
absorbance of light at 260 nm, it is observed that the
absorbance at 260 nm increases as the DNA become
denatured, a phenomenon known as the hyper
chromatic effect or hyperchromacity or
hyperchromism. This is due to un-stacking of base
pairs.
 The rate of increase in absorbance is directly
proportional to the rate of denaturation.
 Viscosity decreases upon denaturation.

12.  It is the formation of base pairs and
complementary strands of DNA come back
together.
 Renaturation occurs if double stranded dna is
heated above Tm then the temperature is slowly
decreased under appropriate conditions.
 Renaturation can be governed by:
 Ionic strength of the solution
 Temperature
 Time
 DNA concentration
 Size of interacting molecule

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