denaturation of dna renaturation of dna

1. DENATURATION
AND
RENATURATION

2. Denaturation and Renaturation
•Heating double stranded DNA can overcome the hydrogen
bonds holding it together and cause the strands to separate
resulting in denaturation of the DNA
•When cooled relatively weak hydrogen bonds between
bases can reform and the DNA renatures
TACTCGACATGCTAGCAC
ATGAGCTGTACGATCGTG
Double stranded DNA
TACTCGACATGCTAGCAC
ATGAGCTGTACGATCGTG
Double stranded DNA
TACTCGACATGCTAGCAC
ATGAGCTGTACGATCGTG
Denatured DNA
Single stranded DNA

3. Denaturation and Renaturation
•DNA with a high guanine and cytosine content has
relatively more hydrogen bonds between strands
• higher GC content is reflected in higher melting or
denaturation temperature
Intermediate melting temperature
Low melting temperature
High melting temperature
67 % GC content -
TGCTCGACGTGCTCG
ACGAGCTGCACGAGC
33 % GC content -
TACTAGACATTCTAG
ATGATCTGTAAGATC
TACTCGACAGGCTAG
ATGAGCTGTCCGATC
50 % GC content -

4. Determination of GC Content
OD260
0
1.0
65 70 75 80 85 90 95
Temperature (oC)
Tm = 85 oC
Tm = 75 oC
Double
stranded DNA
Single
stranded
DNA
Relatively low
GC content
Relatively high
GC content
Tm is the
temperature at
which half the
DNA is melted

5. DNA Renaturation

6. Cot curve
•Cot curve is concerned with the measurement of
the degree of re-annealing of DNA strands.
• It is a curve drawn with X-axis having DNA
concentration unit multiplied by time.
•The initial concentration is considered
represented as Co and when multiplied with time t,
it becomes "Cot" and the graph is known as Cot
curve.
• The graph is drawn against % re-annealed versus
Cot.

7. Reassociation Kinetics
•The following equation describes the second order
rate kinetics of DNA reassociation:
Concentration of single
stranded DNA after
time t
Initial concentration of
single stranded DNA
Second order rate
constant (the
important thing is that
it is a constant)
Co (measured in moles/liter)
x t (seconds). Generally
graphed on a log10 scale.
Cot1/2 is the point at which half
the initial concentration of single
stranded DNA has annealed to
form double-stranded DNA
1
1+kCot
=
C
Co

8. Reassociation Kinetics
Fraction
remaining
single-
stranded
(C/Co)
0
0.5
10-4 10-3 10-2 10-1 1 101 102 103 104
Cot (mole x sec./l)
1.0
Higher Cot1/2
values indicate
greater
genome
complexity
Cot1/2