C0t Curve and its Significance

C0t Curve

The curve obtained by plotting the data of a reassociation kinetics experiment. Since the reassociation of DNA is a bimolecular, second-order reaction, it follows that C/C0=17 (1 + A:C00 where ^_ is the second-order rate constant (L molds’”1), t is the time (s), C0 is the initial concentration of single-stranded DNA (moles of nucleotide per liter), and C is the concentration of single stranded DNA remaining in the reaction mixture at time / (moles of nucleotide per liter).
The cot curve is obtained by plotting the fraction of single-stranded DNA remaining (C/C0) as a function of log (C0Oi that is, the logarithm of the product of the initial concentration and the elapsed time.
The cot curve is an S-shaped curve. See also reassociation kinetics.

Variation in Eukaryotic DNA Sequences –

Prokaryotic and eukaryotic cells differ dramatically in the amount of DNA per cell, a quantity termed an organism’s C value (Table 5.2).
Each cell of a fruit fly, for example, contains 35 times the amount of DNA found in a cell of the bacterium E. coli. In general, eukaryotic cells contain more DNA than that of prokaryotes, but variability in the C values of different eukaryotes is huge.
Human cells contain more than 10 times the amount of DNA found in Drosophila cells, whereas some salamander cells contain 20 times as much DNA as that of human cells.
Clearly, these differences in C value cannot be explained simply by differences in organismal complexity.
So what is all this extra DNA in eukaryotic cells doing?
We do not yet have a complete answer to this question, but examination of DNA sequences has revealed that eukaryotic DNA has complexity that is absent from prokaryotic DNA.


Organism	Approximate Genome Size (bp)
λ -bacteriophage	50,000
E. coli (bacterium)	4,600,000
Saccharomyces cerevisiae (yeast)	13,500,000
Arabidopsis thaliana (plant)	100,000,000
Drosophila melanogaster (insect)	140,000,000
Homo sapiens (human)	3,000,000,000
Zea mays (corn)	4,500,000,000
Amphiuma (salamander)	765,000,000,000

Table 5.2: Genome sizes of various organisms

Denaturation and Renaturation of DNA –

The first clue that the DNA of eukaryotes contains several types of sequences came from the results of studies in which double-stranded DNA was separated and then allowed to reassociate.
When double-stranded DNA in solution is heated, the hydrogen bonds that hold the two strands together are weakened and, with enough heat, the two nucleotide strands separate completely, a process called denaturation or melting (Figure 5.4).
DNA is typically denatured within a narrow temperature range.
The midpoint of this range, the melting temperature (Tm), depends on the base sequence of a particular sample of DNA: G–C base pairs have three hydrogen bonds, whereas A–T base pairs only have two; so the separation of G–C pairs requires more energy than does the separation of A–T pairs.
A DNA molecule with a higher percentage of G–C pairs will therefore have a higher Tm than that of DNA with more A–T pairs.

Figure 5.4: The slow heating of DNA causes the two strands to separate (denature)

The denaturation of DNA by heating is reversible; if single-stranded DNA is slowly cooled, single strands will collide and hydrogen bonds will again form between complementary base pairs, producing double-stranded DNA (Figure 5.4).
This reaction, called renaturation or reannealing, takes place in two steps.
First, single strands in solution collide randomly with their complementary strands. Second, hydrogen bonds form between complementary bases.
Two single-stranded molecules of DNA from different sources will anneal if they are complementary, a process termed hybridization.
For hybridization to take place, the two strands do not have to be complementary at all their bases – just at enough bases to hold the two strands together.
The extent of hybridization can be used to measure the similarity of nucleic acids from two different sources and is a common tool for assessing evolutionary relationships.
The rate at which hybridization takes place also provides information about the sequence complexity of DNA.

Renaturation Reactions and C0t Curves

In a typical renaturation reaction, DNA molecules are first sheared into fragments several hundred base pairs in length. Next, the fragments are heated to about 100° C, which causes the DNA to denature.
The solution is then cooled slowly, and the amount of renaturation is measured by observing optical absorbance.
Double-stranded DNA absorbs less UV light than does single-stranded DNA; so the amount of renaturation can be monitored by shining a UV light through the solution and measuring the amount of the light absorbed.
The amount of renaturation depends on two critical factors:
1) initial concentration of single-stranded DNA (C0) and
(2) amount of time allowed for renaturation (t). Other things being equal, there will be more renaturation at higher concentrations of DNA, because high concentrations increase the likelihood that the two complementary strands will collide.
There will also be more renaturation with increasing time, because there are more opportunities for two complementary sequences to collide.
These two factors together form a parameter called Cot, which equals the initial concentration multiplied by the renaturation time (Co x t = Cot).
A plot of the fraction of single-stranded DNA as a function of Cot during a renaturation reaction is called a C0t curve.
A typical Cot curve for a prokaryotic organism is shown in figure 5.5.

Figure 5.5: A C0t curve represents the fraction of DNA remaining single stranded in a renaturation reaction, plotted as a function of DNA concentration _ time (C0t). This graph is a typical C0t curve for a prokaryotic organism

The upper left-hand side of the curve represents the start of the renaturation reaction, when all of the DNA is single stranded, and so the proportion of single-stranded DNA is 1. As the reaction proceeds, single stranded DNA pairs to form double-stranded DNA, represented by the decreasing fraction of single-stranded DNA. At the end of the reaction, the proportion of single stranded DNA is 0, because all of the DNA is now double stranded.
The value at which half of the DNA is reannealed is called Cot ½.
The rate of renaturation also depends on the size and complexity of the DNA molecules used. Consider the following analogy.
Suppose we distribute 100 cards equally among the students in a class. We ask each student to write his or her name on the cards, and we put all the cards in a hat.
We then randomly draw two cards from the hat and see if the names on the two cards match.
If they don’t match, we put them back in the hat; if they do match, we remove them, and we continue drawing until all the cards have been removed. If there are only four students in the class, each student will receive 25 cards.
Because each student’s name is on 25 cards, the chance of drawing two cards that match is high, and we will quickly empty the hat. If we do the same exercise in another class with 50 students, again using 100 cards, each student’s name will appear on only two cards, and the chance of removing two cards with the same name is much lower. Thus, it will take longer to empty the hat.
This exercise resembles what occurs in the renaturation reaction. If we start with the same total amount of DNA, but there are only a few different sequences in the DNA, a chance collision between two complementary fragments is more likely to occur than if there were many different sequences.
Therefore DNA from organisms with larger genomes will have a larger C0t value.
Thus far, we have considered renaturation reactions in which each DNA sequence is present only once in each molecule.
If some sequences are present in multiple copies, these sequences will be more likely to collide with a complementary copy, and renaturation of these sequences will be rapid.
Think about our analogy of drawing names from a hat. Imagine that we have 50 students and 100 cards; each student gets two cards.
This time, the students write only their first names on the cards. Again, we place the cards in the hat and draw out two cards at random.
If there are five students in the class named Scott, this name will appear on ten cards; so the chance of drawing out two cards at random bearing the name Scott is fairly high. On the other hand, if there is only one Susan in the class, this name will appear on only two cards, and the chance of drawing out two cards with the name Susan is low.
The cards with Scott match up more quickly than the cards with Susan, because there are more copies with the name Scott.
Similarly, in a renaturation reaction, if some sequences of DNA are present in multiple copies, they will renature more quickly.

Types of DNA Sequences in Eukaryotes

For most eukaryotic organisms, C0t curves similar to the one presented in figure 5.6 are produced and indicate that eukaryotic DNA consists of at least three types of sequences. Slowly renaturing DNA consists of sequences that are present only once or at most a few times, in the genome.
This nonrepetitive, unique-sequence DNA includes sequences that code for proteins, as well as a great deal of DNA whose function is unknown.
The more rapidly renaturing DNA represents two kinds of repetitive DNA – DNA sequences that exist in multiple copies. Although not identical, these copies are similar enough to reanneal.

Figure 5.6: A typical C0t curve for a eukaryotic organism contains several steps.

The first step in the curve represents DNA renaturing at very low C0t values, because these sequences are present in many copies (highly repetitive).
The second step represents DNA renaturing at intermediate C0t values; these sequences are present in an intermediate number of copies (moderately repetitive).
The last step represents DNA that renatures slowly; these sequences are present singly or in few copies (unique).
Moderately repetitive DNA typically consists of sequences from 150 to 300 bp in length (although they may be longer) that are repeated many thousands of times. Some of these sequences perform important functions for the cell; for example, the genes for ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) make up a part of the moderately repetitive DNA.
However, much of the moderately repetitive DNA has no known function in the cell.
Moderately repetitive DNA itself is of two types of repeats.
Tandem repeat sequences appear one after another and tend to be clustered at a few locations on the chromosomes.
Interspersed repeat sequences are scattered throughout the genome.
An example of an interspersed repeat is the Alu sequence, each of which consists of about 200 bp.
The Alu sequence is present more than a million times in the human genome and makes up about 11% of each person’s DNA. Short repeats, such as the Alu sequences, are called SINEs (short interspersed elements).
Longer interspersed repeats consisting of several thousand base pairs are called LINEs (long interspersed elements).
Most interspersed repeats are transposable genetic elements, sequences that can multiply and move.
The other major class of repetitive DNA is highly repetitive DNA.
These short sequences, often less than 10 bp in length, are present in hundreds of thousands to millions of copies that are repeated in tandem and clustered in certain regions of the chromosome, especially at centromeres and telomeres.
Highly repetitive DNA is sometimes called satellite DNA, because it has a different base composition from those of the other DNA sequences and separates as a satellite fraction when centrifuged at high speeds.
Highly repetitive DNA is rarely transcribed into RNA.
Although these sequences may contribute to centromere and telomere function, most highly repetitive DNA has no known function.