respectively. The mixture was incubated at 37°C for 60 min, made 150 g/ml Proteinase K (Boerhinger Mannheim),
and incubated at 37°C for an additional 60 min. Two phenol/chloroform/isoamyl alcohol extractions were then
performed followed by an extraction using only chloroform. The final aqueous phase was transferred to a small
siliconized beaker, and 3M sodium acetate (pH 5.2, 4°C) was mixed with the DNA solution to give a final sodium
acetate concentration of 0.15 M. Two volumes of 100% ethanol (-20°C) were layered on top of the nucleic acid
solution, and DNA was spooled from the interface using a glass rod. The DNA was dried, redissolved in a small
volume of TE buffer (pH 7,0), and stored at -20°C.
Approximately 2-3 mg of DNA was obtained from 300 g of seedlings or 400 g of leaf tissue. The DNA
possessed no visible coloration. Isolated nuclear DNA had A260 /A280 ratios (adjusted for light scatter at A320) from
1.8-1.93 suggesting little protein contamination. The mean length of the DNA molecules was >13 Kb as
determined by agarose gel electrophoresis. No RNA contamination was observed. The isolated tomato DNA was
completely digestible with 1 U of Hindlll or Baml-H1 per µ g of DNA. The tomato DNA possessed normal melting
and reassociation properties (see accompanying papers).
Acknowledgments:
The authors thank Kevin Boehm for technical assistance. This work was funded in part by U.S.D.A. Grant Award
No. 95-37300-1570.
Literature cited:
Couch, J.A., and Fritz, P.J. (1990) Plant Mol. Biol. Rep. 8: 8-12.
Guillemaut, P. and Marechal-Drouard, L. (1992) Plant Mol. Biol. Rep. 10: 60-65.
Katterman, F.R.H. and Shattuck, V.I. (1983) Preparative Biochem. 13: 347-359.
Watson, J.C. and Thompson, W. F. (1986) Meth. Enzymology 118: 57-79.
Characterization of the tomato genome using Cot analysis
Peterson, D.G. and Stack, S.M.
Department of Biology, Colorado State University, Fort Collins, CO 80523.
Cot curves can be used to determine (1) genome size (= 1C DNA amount), (2) the fraction of a genome
composed of single-copy sequences, (3) the minimum number of repetitive classes of DNA, (4) the fraction of a
genome occupied by each repetitive class, (5) the number of repeats in each repetitive class, and (6) the
sequence complexity of all curve components (see Britten et al. 1974 for review). To prepare a Cot curve,
samples of sheared nuclear DNA are dissolved in sodium phosphate buffer, heat denatured, and then incubated
at a renaturing temperature for different lengths of time. DNA sequences reassociate at a rate that is directly
proportional to the number of times they occur in the genome. Thus sequences that occur more than once
(repeated sequences) reassociate faster than sequences that occur only once in the genome (single-copy
sequences). Under standard conditions, the Cot value of a particular sample is the product of nucleotide
concentration (moles/L) and the incubation time in seconds (Britten et al. 1974). Once a sample has reached a
desired Cot value, it is loaded onto a hydroxyapatite (HAP) column. The single-stranded (non-renatured) and
double-stranded (renatured) DNA are separately eluted from the column by heating the column or by increasing
the cation concentration in the column. If the logarithm of a Cot value is graphed against the corresponding
fraction of the genome that has remained single-stranded, the resulting data point is called a Cot point. A graph of
Cot points ranging from little or no reassociation (low Cot points) until reassociation approaches completion (high
Cot points) is called a Cot curve.
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