OVERVIEW OF COT ANALYSIS

For several years now, the term “genomics” has been a “buzz word” in the scientific community.  However, the first detailed, comparative investigations of genomes (arguably the first genomic experiments) were performed roughly 35 years before scientists, academic departments, and scientific institutes started using the “genomics” moniker.  In the 1960s, Roy Britten and his colleagues began investigating the renaturation kinetics of genomic DNA in solution using a technique they called “Cot analysis”.  In a Cot analysis, the compound hydroxyapatite (HAP) is used to preferentially separate renatured (double-stranded) DNA from single-stranded DNA.  An overview of the steps in a Cot analysis is shown in FIGURE 1.  From a Cot analysis one can determine genome size, the fraction of single-copy DNA in a genome, and the number, size, complexity, and relative arrangement (dispersed vs. tandem arrays) of repetition-based DNA components.  Likewise, Cot analysis allows many of the basic characteristics of genomes to be compared between genetically similar/dissimilar organisms.  With the advent of molecular cloning techniques, most genome researchers abandoned the notoriously difficult business of Cot analysis.  However, the principles of nucleic acid hybridization developed through Cot research form the basis of many molecular biology techniques, and information generated in Cot studies remains central to current knowledge of genome structure.

 

FIGURE 1. Overview of Steps Involved in a Cot Analysis.  (A) Plant leaves are placed in an antioxidant medium and homogenized in a blender.  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 by phase-contrast microscopy).  DNA is isolated from purified nuclei using phenol/chloroform extractions coupled with proteinase and RNase digestions.  (C) The DNA is cut into pieces between 200-500 bp by 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 sodium phosphate buffer (SPB) to produce solutions of known concentrations.  (E) The DNA solutions are distributed into glass microcapillary tubes or glass ampoules so that each tube/ampoule contains 100 µg of DNA.  The ends of the tubes/ampoules are sealed.  One of the tubes (F) containing a known concentration of DNA is placed in boiling water to denature DNA duplexes (G).  (H) The tube then placed in a water bath set at a defined temperature.  Renaturation is allowed to occur until the sample reaches a specific Cot value [Cot value = the product of the sample's nucleotide concentration (moles of nucleotides per liter), its reassociation time in seconds, and an appropriate buffer factor based upon cation concentration].  (I) Once the sample has reached the desired Cot value, the end is broken off of the tube/ampoule, and the solution is blown into a 100-fold excess of 0.03 M SPB.  (J) The diluted sample quickly is loaded onto a hydroxyapatite (HAP) column equilibrated with 0.03 M SPB.  At this buffer concentration, all DNA binds to the HAP.  (K) Once all of the solution has entered the HAP, 0.12 M SPB is added causing single-stranded DNA (ssDNA) to elute.  Eluant containing ssDNA is collected in a graduated polypropylene tube.  (L) After the ssDNA had been collected, 0.50 M SPB is added to the column to elute double-stranded DNA (dsDNA).  (M) The volumes of the ssDNA eluant and the dsDNA eluant are determined.  (N) Exactly 0.9 ml of the centrifuged ssDNA eluant is mixed with 0.1 ml of aqueous 10 N KOH to denature any DNA duplexes.  A sample of the dsDNA eluant is likewise denatured.  (O) The A260 values (adjusted for light scatter at 320 nm) of the ssDNA/KOH mixture and the dsDNA/KOH mixture are determined.  For a particular Cot value, the percentage of ssDNA (% ssDNA) is calculated as follows:

[(Vss x Ass) x 100] ÷ [(Vss x Ass) + (Vds x Ads)] = % ssDNA

where Vss = total volume of single-strand fraction, Vds = total volume of double-strand fraction, Ass = A260 (adjusted for light scatter) for the KOH-denatured single-strand fraction, and Ads = A260 (adjusted for light scatter) for the KOH-denatured double-strand fraction.  (P) The logarithms of Cot values ranging from essentially no renaturation to nearly complete renaturation are plotted against corresponding % ssDNA values to yield Cot points.  A graph of Cot points ranging from little or no reassociation until reassociation approaches completion is called a Cot curve.  Because a DNA sequence reassociates at a rate that is directly proportional to the number of times it occurs in the genome, sequences that occur more than once in a genome (repetitive DNA) reassociate at lower Cot values than sequences found only once per genome (single-copy DNA).  From an analysis of a Cot curve one can determine genome size, relative proportions of single-copy and repetitive sequences, the fraction of the genome occupied by each frequency component, and the complexity of the sequences in each frequency component.