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In the case of denaturation and renaturation of DNA fragments can be affected by the application of heat. Cot value signifies the concentration of denatured DNA at C1/2 value. Presence of buffer factors including the cations (+) also affects the cot value. What is the Cot value analysis? How does heat affect the Cot curve? Those questions have to be discussed to analyze the Cot value curve. This study also discusses the effect of buffers including cations and the concentration of single stranded DNA after a time interval. Denaturation means breakdown of double stranded DNA by application of heat and when heat is removed from the medium DNA strand, again starts to reanneal.
Cot value
The Cot value signifies the DNA concentration while multiplied with time and buffer factors in a medium. Cot value may be affected by the concentration of the DNA bases in a double stranded DNA. Denaturation time of a double stranded DNA also depends on the N2 bases present in a strand. If the concentration of Guanine (G) and Cytosine (C) is more in a double stranded DNA, then it will take more time to denature. Guanine and Cytosis bind with each other by forming a triple bond while Adenine and Thymine added with a double bond. When heat is removed from the medium DNA bases start to renneal. Cot value can be calculated by multiplying the factors including concentration of DNA with time and buffer factors.
Cot curve
Value of Cot curve signifies the 50% denaturation of a DNA sample that should be affected by the application of heat and buffer concentration. In the case of Cot curve time is measured by a time unit of seconds. In the Cot curve, the X-axis signifies the concentration of DNA at the initial point and the Y-axis signifies the percentage of single stranded DNA. The concentration of DNA determines the value Cot, if the DNA concentration is high then the Cot value will be high. The viscosity of the medium also can affect the graph of the Cot value. If the DNA sample has some impurities then the viscosity will change. Otherwise, in case of a pure DNA sample the viscosity does not matter at all.
Percentage of single stranded DNA
In the Cot value curve Y-axis determines the concentration of the single stranded DNA. In order to determine the Cot value curve, concentration of the single stranded is a major factor to determine the value of the curve. Double stranded DNA strands are denatured by applying heat in the media and form single stranded (SS) DNA. In order to calculate the Cot value, concentration of SS helps to determine the genome complexity of the sample DNA strand. When heat is removed from the medium SS DNA starts to rejoin and again form the double stranded DNA.
Time for Cot value curve
In order to calculate the Cot value, time of denaturation and re-anneal have a great impact over the curve value. In the case of high numbers of N2 bases including Adenine (A) and Guanine (G), time for denaturation takes more time because A binds with C by forming a triple bond. The same thing is going for the re-anneal, as the number of a specific gene sequence is present in a high concentration it will take more time to re-anneal. Measurement of the time also helps to determine the complexity of the sample DNA strand.
Buffer factors
Buffers help to maintain the pH value of targeted samples. Natural pH value of DNA lies between 5 to 9. In case of higher pH value, DNA faces destabilisation. In case of determining the value of Cot curve, buffer factors help to maintain the concentration of anion and cations of the reaction.
Application of Cot curve
Cot value curve helps to determine the complexity of a specific gene sequence in a sample DNA strand. This curve helps to determine the number of repetitive sequences of a DNA strand. In the case of highly repetitive DNA bases the curve will show a high growth rate, in case of moderate repetitive sequence the re-anneal graph shows a moderate pick. In the case of a low number of repetitive sequences, the graph provides a low pick that helps to analyse the complexity of the sample DNA strand. Value of the Cot curve also helps to determine the structure of the genome and it helps to simplify the DNA sample.
Conclusion
The Cot value helps to determine the complexity of the sample DNA. This curve value also helps to find the number of unique sequences present in the sample DNA.
