Cot Curve Analysis (2024)

Table of Contents

• Concept of Denaturation and Renaturation of DNA
• What is a C0t Curve?
• Procedure of Analysis
• Analysis of C0t Curve
• C0t Curve and its Significance

C₀t analysis is a biochemical technique that is used to find out the measure of repetitive DNA in a DNA sample. It is based on the principle of DNA renaturation. It is a technique that comes in handy while studying a genome and simplifying it by finding out the large amounts of repetitive sequence it contains.

Concept of Denaturation and Renaturation of DNA

The breaking down of the hydrogen bonds of the DNA strands such that it gives rise to two single DNA strands is known as denaturation of DNA. Conditions such as high temperature, high pH and presence of denaturing agents can lead to the breakdown of hydrogen bonds.

When the extreme temperature and pH conditions return to normal, the DNA starts to re-anneal to form double stranded DNA, this is known as DNA renaturation or DNA annealing.

Refer: Nucleic Acids – Functions, RNA & DNA Structure

What is a C₀t Curve?

While plotting a graph for the renaturation of DNA, the Y-axis is marked as the percentage of DNA that remains single. The percentage is calculated as a ratio of the concentration of ssDNA (C) by the total concentration of DNA (C₀).

The X-axis is a product of the total or initial concentration of DNA (C₀) and the time taken for the reaction in seconds (t). This value comes out as C₀t, hence the graph is also known as the C₀t curve. While calculating the C₀t value, a buffer factor that accounts for the effect of cation is also taken into consideration.

Procedure of Analysis

• Heat the genomic DNA sample until it denatures and forms two single stranded DNA.
• Slowly cool down the temperature so that the DNA starts to anneal.
• Take samples at different temperatures while the DNA is annealing.
• Determine the C₀t value of the genomic sample by plotting the above mentioned graph.

Analysis of C₀t Curve

• The rate of renaturation of DNA depends upon the concentration of DNA, temperature of reassociation, concentration of cation and viscosity.
• A sequence with higher number of repetitive DNA will renature at a faster rate than the one which has a lower number of repetitive DNA.
• If given enough time, all of the DNA might renature in a given sample.
• A low C₀t value indicates that the sequence has a larger number of repetitive sequences.
• On the other hand, a high C₀t value indicates that the genomic sample is unique, i.e., it has a low number of repetitive sequences.

C₀t Curve and its Significance

The C₀t analysis acts as a filtration technique that filters out large volumes of repetitive sequences from the complex eukaryotic genome such that only unique, low copy number sequences are left. This makes the study of these unique sequences easy and thus greater information can be extracted about the genome.

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Also Read:

• Difference between Denaturation and Renaturation
• Introduction and Differences between DNA and RNA
• Double Helix Structure of DNA
• Histones & Importance of DNA Packaging
• How did DNA Evolve? – Evolution and Discovery of DNA