Cot Analisys

Cot Analysis: An Overview

Cot analysis was first developed and utilized in the mid 1960s by Roy Britten, Eric Davidson, and associates. It is based upon the principles of DNA renaturationkinetics.

DNA Renaturation Kinetics • The rate at which heat-denatured DNA sequences in solution will renature is dependent on DNA concentration, reassociation temperature, cation concentration, andviscosity (usually not a factor if the DNA is free of contaminants). • Cot = DNA conc. (mol/L) x renaturation time in sec x a buffer factor that accounts for the effect of cations on the speed ofrenaturation

The same Cot value can be reached in a variety of ways For example: Nucleotide concentration Renaturation time Buffer factor, 0.5 M SPB

= = X =

0.050 M 344.000 sec 5.820 Cot 100.000Nucleotide concentration = Renaturation time = X Buffer factor, 0.12 M SPB =

0.002 M 50,000.000 sec 1.000 Cot 100.000

The total amount of reassociation for a given Cot value does not change.• The rate at which a particular sequence will reassociate is proportional to the number of times it is found in the genome. • Given enough time, nearly all of the DNA in a heat-denatured DNA samplewill reassociate.

The steps in a Cot analysis

A

B

Nuclei stained with methylene blue

(A) Tissue from the organism of interest is placed in an antioxidant medium and homogenized in ablender. The homogenate is filtered, plastids are preferentially lysed, and nuclei are pelleted by centrifugation. (B) The pellet should be free of contaminating organelles (as determined byphase-contrast microscopy). DNA is isolated from purified nuclei using phenol/chloroform extractions coupled with proteinase and RNase digestions.

C

D

E

(C) The DNA is cut into pieces of about 450bp by sonication or high-speed blending. (D) Fragment size is checked by agarose gel electrophoresis. Sheared DNA is precipitated, and aliquots of the DNA are dissolved in 0.03 M, 0.12 M, and 0.50 M...