MEASURING GENOME SIZE AND COMPLEXITY USING C₀T CURVES

Rate of reassociation

1. Shear DNA to ca. 400bp (eg. blender,sonicator, passage through a small orifice).
2. Denature fragments by boiling.
3. Reassociate in 0.18M salt
4. Measure A₂₆₀ or, if radioactively labeled, amount bound to HAP (hydroxyapatite) column. HAP preferentially binds dsDNA at 0.18M salt

The reassociation of DNA molecules in solution is described by the relation:

        kC2
 -dC  = ---
        dt

C  ::= [ssDNA] (mole L-1)

dC ::= change in [ssDNA]/sec.

t  ::= time (sec.)

k  ::= 2nd order rate constant, dependent on temperature and
       ionic conditions and complexity of DNA (L mole-1 sec-1)

 C         1
---  = -----------
 C0     1 + k C0t

C₀ ::= the initial[ssDNA] at time 0.
C₀t_½ ::= the value of C₀t at which annealing has proceeded to half completion (C/C₀=0.5).

Redrawn from Russel, P.J. (1986) Genetics Figure 7.24. Ideal time course for the renaturation of DNA as seen in a C_ot (initial DNA concentration x time) plot. In the initial state the DNA is single-stranded, and in the final state it is all double-stranded. Note that 80 percent of the renaturation occurs over a 2 log C_ot interval.
 

Complexity

Demo: kinetic_class_demo.obj

The complexity (X) of a population of uniformly-sized DNA molecules can be measured as follows:

X= KC₀t_½

where K has been determined under standard conditions (0.18M cations {eg. Na ⁺ }, 400 nucleotide fragmentsize) as approximately 5 x 10 ⁵ L bp mole^-1 sec^-1 .

Redrawn from  Russel,P.J. (1986)Genetics Fig. 7-25a. C_ot plots showing the renaturation of DNAs from organisms with small genomes: the bacterium E.coli, the bacterial viruses T2 and Lambda, and the animal virus SV40.

Redrawn from  Russel,P.J. (1986 )Genetics Fig. 7-25b. Kinetics of renaturation of DNAfrom calf thymus and E. coli as seen in a Cot plot. The E.coli DNA consists almost entirely of unique sequences. However, the shape of the Cot curve for the calf DNA is very different from that of E. coli and indicates that there are some sequences (toward the right of the curve) that renature much more slowly and some (toward the left of the curve) that renature much more quickly than the bacterial DNA sequences.

 

The C₀t curves for Calf thymus and E.coli DNA indicate that, while E.coli DNA anneals at a relatively sharp inflection point, Calf DNA contains three major kinetic classes: highly repetitive, which reanneals very early in the reaction, middle repetitive, which anneals over more than 3 log C₀t, and single copy, which anneals at very high C ₀ t values.

The term "single-copy" is a bit misleading, in that it refers to 1 - 10 copies per haploid genome. In fact, any distinction between single copy and middle repetitive  forces you to draw an arbitrary line. They are useful concepts because they bring out something of the content of genomes, but the definitions of highly repetitive,  middle repetitive and single-copy shouldn't be pushed too far.

Another point to mention is that although most protein coding genes are found in the single copy fraction,  not all of the single copy fraction is protein coding genes. There appears to be a lot of non-coding single copy "junk" in many eukaryotic genomes.

Calculation of complexity of a single kinetic class

C0t½ (pure) = fC0t½ (mixture)

X = KC₀t_{½ (pure)}

Table 1 from Okamuro & Goldberg p8 in  The Biochemistry of Plants Vol. 15

Databaseof Genome Sizes (http://www.cbs.dtu.dk/databases/DOGS/)"

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