Single-stranded nucleic acid polymers that possess complementary base sequences can combine to form duplexes when mixed under appropriate solution conditions. For complementary short oligonucleotides, the reassociation process is fast and complete. For complementary polymeric nucleic acids, the annealing process is more complex, slower, and not necessarily complete. Monitoring of the rate of reassociation of the duplex is a classical method for evaluating the relative complexity of genomic DNA. The duplex DNA is fragmented by exposure to shear gradients or restriction enzymes. Fragmentation is followed by thermal denaturation to produce single-stranded fragments. The reassociation process is monitored as a function of time. From these data, plots are constructed of the fractional extent of reassociation as a function of C^t, where C^ is the initial nucleotide concentration and t is time. The value of C^t at which the fractional extent of reassociation is 0.5, is a measure of the complexity of the genome (see C0t Curve).
Double-stranded DNA exhibits hypochromicity; the absorbance of the duplex is less than the sum of the absorbance of the constituent single strands. Changes in absorbance on reassociation to form duplex DNA can be used to estimate the extent of reassociation at any point in time.
Under conditions of low sodium phosphate concentration, both single-stranded and double-stranded DNA will bind to hydroxyapatite. At moderate sodium phosphate concentrations, 0.1-0.2 M, double-stranded DNA is retained on the column, but single-stranded DNA is eluted. Double-stranded DNA is eluted at 0.5 M sodium phosphate concentration. Because double-stranded and single-stranded DNA can be discriminated by their retention on the hydroxyapatite column, the time course for the reassociation process can be monitored.
