Limitations of the EST Data

Figure A Overview of Steps Involved in a Cot Analysis. (A) Nuclear DNA is sheared into 200-500 bp fragments by high-speed blending, and (B) fragment size is checked by gel electrophoresis. (C) Sheared DNA is precipitated, dissolved in 0.03, 0.12, or 0.5 M sodium phosphate buffer (SPB), and aliquots are loaded/sealed into glass microcapillary tubes/ampoules. (D) One tube/ampoule is (E) placed in boiling water to denature DNA duplexes, and then transferred (F) into a water bath set at the DNA sample’s “criterion”, i.e., Tm 25oC for genomic DNA in the same SPB buffer as the sample. Renaturation is allowed to proceed to a specific Cot value (Cot = the product of nucleotide concentration, reassociation time, and an appropriate factor based upon the cation concentration of the buffer). (G) Once the sample has reached the desired Cot value, it is quickly diluted in a 100fold excess of 0.03 M SPB, and (H) loaded onto a hydroxyapatite (HAP) column equilibrated with 0.03 M SPB. At this buffer concentration all DNA binds to the HAP. (I) 0.12 M SPB is added causing single-stranded DNA (ssDNA) to elute. (J) 0.50 M SPB is added to the column to elute double-stranded DNA (dsDNA). (K) The volumes and (L) A260 values (adjusted for light scatter at 320 nm) of the ssDNA eluant and the dsDNA eluant are used to determine the percentage of ssDNA (% ssDNA) for the Cot value (see Peterson et al. 1998 for details). Steps D-L are repeated for all the DNA samples with each sample being renatured to a different Cot value. (M) The logarithms of Cot values ranging from essentially no renaturation to nearly complete renaturation are plotted against corresponding % ssDNA values to yield 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, repetitive sequences reassociate at lower Cot values than single/low-copy sequences. Analysis of a Cot curve permits estimation of genome size, the number and relative proportion of the repetitive and single/low-copy components comprising the genome, and each component’s ‘kinetic complexity’ (i.e., its estimated sequence complexity). D B C