Abhiram krishnan p central university of kerala

1. Cot curve and Tm
Abhiram Krishnan p
I M.sc. Zoology
Central university of Kerala

2. Absorption of uv light
 Nucleic acid exhibits characteristic absorption in the ultraviolet region, this is due to the conjugated
double bonds and the ring system of constituent purine and pyrimidine.
 The more ordered the structure, less light is absorbed.
 Free nucleotide absorb more light than single-stranded polymer of DNA & RNA and these inturn
absorbes more light than a double-stranded DNA molecule.
 The maximum absorption is at 260nm and minimum at 230nm.
 Absorption is proportional to the concentration of the molecule, with a value of 0.02 units per µg
DNA per ml.
 Double stranded DNA is said to be hypochromic & bases are hyperchromic.

3.  Example;
3 solutions of double stranded DNA, single stranded DNA and free bases each at 50µg/ml have the following A260
values.
 DS DNA =1.00
 SS DNA = 1.34
 Free base = 1.60
 DENATURATION OF DNA MOLECULES
 The ordered state of DNA helix, which is orginally present in nature is called the native form.
 The two strands of DNA readily come apart when the hydrogen bonds b/w its paired base 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 translation from the native to the denatured state is called denaturation.
 Denaturation of DNA can be studied by measuring its absorbance at a wavelength of 260nm.
 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 DNA molecules.
 The temperature at which half of the helical structure of DNA molecules is lost is called its melting
temperature(Tm). A convenient parameter to analyse melting transition.
 Molecules rich in GC pair have a higher Tm than those having abundance of AT base pairs because of more
stability & held together by three hydrogen bonds. Such DNA molecules require more energy and hence
temperature to denature.

4. Denaturation involve changes
 Denaturation converts the firm, helical two-stranded native structure of DNA to a flexible,
single-stranded denatured state.
 The splitting of DNA molecules 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.
 Increase in absorption of UV light: Due to increase, all of the bases in nucleic acid
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 coin) 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 posses a high viscosity because of the
relatively rigid double helical structure and long, rod-like character of DNA. Disruption of the
hydrogen bonds causes a marked decrease in viscosity.

5. TM curve
Denaturation and absorbance
 For example(the absorption of ultraviolet light), if a solution of double-stranded DNA has avalue 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 hyperchromatic
when heated.
 The following features of this curve should be noted:
 A260 remains constant upto temperatures well above those encountered by most living cells in
nature.
 The rise in A260 occurs over a relatively narrow range of 6-8 degree Celsius.
 The maximum A260 is about 37% higher than the starting value.

6. How to denature DNA
 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.The phenomenon is called breathing.
Which enables specialized proteins to interact with DNA molecules and to read its encoded
information.
 Breathing occurs more often in the 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 prefers 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 treating with alkali. Since DNA is quite
resisitant to alkali hydrolysis, this procedure is the method of choice for denaturing DNA,
because that treatment may often break the phosphodiester bonds and may result in
yielding broken fragmets of DNA.

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 os 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.
 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 pairing 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.

9. Renaturation rate and DNA sequence complexity
COT CURVE
 The renaturation rate of DNA is an excellent indicator of the sequence complexity and the
sixe of the DNA.
 Eg; Bacteriophage T4 DNA contains about 2x105 nucleotide pairs, where as Escherichia coli
DNA possesses 4.64x105. 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 gm), the sequences
represented in an E, coli sample are more heterogenous, 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
analysed quantitatively.
 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
when 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 unites of C are moles of ntd per L and t is in second.
 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,

10. 0.5 = 1/(1+k2C0t1/2) and thus 1 + k2C0t1/2 comes out to be 2
yielding C0t1/2 = 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.