section 23.4
Renaturation of DNA
529
2. The temperature must be high enough to disrupt the
random, intrastrand hydrogen bonds described above.
However, if the temperature is too high, stable
interstrand base pairing will not occur or be
maintained. The optimal temperature for renaturation
is 20-25°C below the value of
Tm.
Renaturation is slow compared with denaturation. The
rate-limiting step is not the rewinding of the helix but the
precise collision between complementary strands such that
base pairs are formed at the correct positions. Since two
molecules participate in the rate-limiting step, renatura-
tion is a concentration-dependent process requiring several
hours under typical laboratory conditions. In particular,
the kinetics of association follows a simple second-order
rate law with the association rate increasing with DNA
concentration. The kinetics are conveniently described by
an equation that relates the fraction, / , of single-stranded
(dissociated) DNA and the time elapsed after exposing a
DNA sample to renaturing conditions (high salt concen-
tration, elevated temperature):
/ =
1/(1
+
kCot)
in which Co is the initial DNA concentration,
t
is the
elapsed time in seconds, and
k
is a rate constant. A plot
of / versus the logarithm of the product
C0t
yields a
sigmoid curve commonly called a
Cot
(“cot”) curve. A
set of such curves for several DNA samples is shown in
Figure 23-9. A notable feature of these curves is that the
renaturation rate is related to the molecular weight of the
DNA. A useful index for characterizing these curves is
Colti/
2
,
with the value of
Cot
corresponding to renatura-
tion of half of the DNA ( / = 1/2). Comparison of the
Colt
\ / 2
values for
E. coli
DNA (M.W. = 2.7 x 109) and
T
4
DNA (M.W. = 1.1 x 108) shows that T
4
DNA rena-
tures roughly 50 times faster than
E. coli
DNA. The reason
FIGURE 23-9
Reassociation
“C ot”
curves for bacteriophage T4 DNA,
E. coli
DNA, and
mouse DNA. [Reproduced with permission from D. M. Prescott:
Cells:
P rinciples o f M olecular Structure and Function.
Jones and Bartlett,
London, 1988.]
FIGURE 23-10
(a) Hypothetical DNA molecule having a base sequence that is 3% of the
total length of the DNA and is repeated five times. The dashed lines
represent the nonrepetitive sequences: they account for 85% of the total
length, (b) Renaturation curve for the DNA in (a). Time is logarithmic to
keep the curve on the page.
is that if the two DNA samples have equal molar concen-
trations of nucleotides, the T
4
sample will contain many
more DNA molecules than the
E. coli
sample.
Cot
analysis is not -usually done with intact DNA
molecules but rather with fragments having lower molec-
ular weights. This breakage does not affect the relative
values of
Colt
]/2
since the number of different kinds of
fragments of T
4
DNA is smaller and hence their concen-
tration larger than the corresponding number of fragments
in the
E. coli
sample. Studies of a variety of prokaryotic
DNAs show that the value of
Colt
]/2
is directly related to
the total size of the DNA of the organism (the genome
size). However, this relation does not apply to the DNA
of eukaryotes because of the presence of highly repetitive
sequences.
The fragmentation of the DNA molecules allows a new
feature of base sequences to be seen. Figure 23-10(a)
shows a long hypothetical DNA molecule having a repeat-
ing base sequence. If the unbroken denatured DNA were
allowed to renature, the
Cot
curve would be a smooth curve
in which Co/
0 / 2
would be proportional to the size of the
DNA. However, if the DNA were broken into small pieces,
several fragments from each molecule would contain the
repeated sequence, and these fragments would renature
more rapidly than the bulk of the sequences. For example,
if the repeated sequence contains 3% of the total num-
ber of bases of the DNA and there are five copies of the
repeated sequence in the DNA, the
Cot
curve would be
that shown in Figure 23-10b, in which the more rapidly
renaturing component accounts for 15% of the transition;
hence, the conclusion that 15% of the DNA (on a weight
basis) contains a repeating sequence. Approximately 30%
of human DNA contains sequences of bases that are re-
peated
2 0
times or more.
