Cot Curve of DNA and its Significance

Cot curve is a technique used to measure the complexity of DNA or a genome. The DNA renaturation is analysed by this method.

When the extent of renaturation of DNA is plotted against log Cot , this is called Cot curve.

The cot curve technique was discovered by Roy Britten and Eric Davidson in 1960. This process is based on the principle of DNA renaturation kinetics.

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Cot curve of DNA

Genome complexity is a total length of different sequences of DNA. It can be measured by the renaturation kinetics of denatured DNA. Renaturation of DNA occurs because of complementary base pairing of DNA strands. Renaturation depends upon random collision of the complementary DNA strands.

For plotting the graph of renaturation of the DNA, the Y axis of the graph is taken as the percentage of DNA that remains single.

This percentage of DNA is a ratio of Concentration (Co) of single stranded DNA (ssDNA) and the total concentration of DNA .

Along the X axis the product of initial concentration of DNA and the time (in second) taken for the reaction is taken. After plotting the X and Y axis the value comes out as Cot. So the curve is also called the Cot curve of DNA. At the time of calculating the Cot value, a buffer factor that accounts for the effect of cation is also taken into consideration.

DNA denaturation cot curve

Cot curve principle

The rate of renaturation of DNA is directly proportional to the number of times the sequences are present in the genome.
Enough time is given so that all DNA that is denatured will reassociate or reanneal in a given DNA sample.

The more the repetitive sequence the less will the time take for DNA renaturation.

The rate of renaturation is directly proportional to the time. The rate of renaturation can be followed spectrophotometrically by the UV absorbance decreases as the duplex DNA is formed. So a Cot curve describes the kinetics of renaturation of a sample of ds DNA that has been fragmented into small pieces, denatured to single strand and then permitted to reanneal.

Common sequences reanneal more rapidly than rare sequences. Indeed the rate at which sequence will reassociate is proportional to the number of copies of that sequence in DNA sample. A simple with highly repetitive sequences DNA will renature rapidly, while complex sequences will renature slowly.

The renaturation of DNA depends on the following factors –

DNA concentration
Renaturation time
Temperature
Buffer factor
Viscosity

Read more from us in the topic Polymerase Chain Reaction

Cot value / Cot Equation

However, instead of simply measuring the percentage of dsDNA vs time, the amount of renaturation is measured relative to the cot value. Repetitive DNA will renature at lower Cot Value. While complex DNA sequences renature at higher cot values.

Degree of Renaturation after given time depends on Co and t.

Cot value = Co × t × buffer factor

Cot value equation

Meaning of Co, t and buffer factor in cot equation

Co stands for nucleotide concentration in moles per liter prior to denaturation.

t is the reassociation time in seconds.

buffer factor is a factor based upon the cation concentration of the buffer.

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COT Analysis

The genomic DNA is first cut randomly into fragments about 1kb in length.
Fragments are completely denatured by heating above the melting temperature. (Tm =temperature at which 50% of the DNA is melted)
Fragments are allowed to renature(renaturation at a temperature about 10°C below the Tm).

Renaturation at different time intervals can be monitored by two ways:

using Spectrophotometer(by taking absorbance at 260nm- as ssDNA absorbs light more efficiently than ds DNA (due to base stacking it has more constrained bases).
using Hydroxylapitite chromatography(HC)- an adsorption chromatography, Hydroxylapitite (modified form of crystalline calcium phosphate) adsorb double stranded DNA as the positively charged calcium in hydroxylapitite will have more affinity towards double stranded DNA as dsDNA consists of more negatively charged phosphate groups than ssDNA.

FAQ related to Cot curve

What is the use of Cot curve analysis?

Analysis of a Cot curve gives data about genome size, the proportion of the genome contained in the single-copy and repetitive DNA components, and the kinetic complexity of DNA.

What is cot value and what does it measure?

Degree of Renaturation after given time depends on Co and t. Cot value = Co × t × buffer factor. Co = nucleotide concentration in moles per liter prior to denaturation. t = reassociation time in seconds. buffer factor= a factor based upon the cation concentration of the buffer.

What is Cot curves of DNA?