Molecular Genetics

The kinetics of DNA reassociation reveal DNA classes differing in repetition frequency.

Facts

• DNA is sheared to fragments of 300 bp average length. The DNA is denatured.
• Various concentrations of the DNA are incubated for varying times at a temperature allowing reassociation.
• The fraction of the DNA not reassociated in each sample is measured and plotted.

• Hydroxylapatite (HAP) is used to distinguish single-stranded from double-stranded DNA. All DNA is bound at low salt concentrations. At intermediate salt concentrations, partially or completely double-stranded DNA remains bound to HAP, while single-stranded DNA elutes. Top
• C_ot
  • C_ois the concentration of single-stranded DNA at the beginning of the reassociation reaction. It is measured in moles of nucleotide units per liter.
  • t is the time of the reassociation reaction, in seconds.
  • The amount of single-stranded DNA left (C/C_o) is plotted against the product of the above two terms, C_ot. C_ot is used because it appears in the equation describing reassociation kinetics:

    1 C/Co = ----------------- 1 +k Cot

  • When the DNA is 50% reassociated:
    0.5 = 1 / (1+ k C_ot_1/2)
    So, C_ot_1/2 = 1 / k
  • C_ot_1/2 and k (reassociation rate constant) describe the kinetic behavior of DNA reassociation. Top
    
    
• Fold-back (or snap-back) DNA renatures without incubation due to the presence of inverted repeats. The lengths of duplexes formed range from 200 to 1200 bp; some are as large as 6 kbp (see Table). Fold-back DNA can consist of double-stranded rods or stem-loop structures. Non-contiguous inverted repeats form loops:
  ----------> ..........<-----------
  The fraction of the genome that forms foldback DNA ranges from 1 to 30% (see Table). Some fold back DNA is due to the chance proximity of stretches of repeated DNA. In Drosophila, much of the fold back DNA is due to the FB transposable element. Fold-back DNA has not been detected in bacteria. Top

• Highly repeated DNA generally has a C_ot_1/2 < 10^-2. It accounts for 1% to >15% of the genome, with most genomes in the 10-15% range. Highly repeated DNA is located near centromeres, near telomeres, and is associated with heterochromatin. In some organisms, it may also be interspersed in gene-containing regions. Top

• Moderately repetitive DNA consists of families of related sequences, not necessarily identical to one another. The stringency of reannealing allows some mismatched base pairs to form. Thus, renatured moderately repetitive DNA melts with a lower T_m than renatured unique DNA or native DNA. Some moderately repetitive DNA families represent multi-copy structural genes. Note that in some organisms (see Table), the moderately repetitive class can be split into more than one group. Actually, there may be a continuum of frequencies of repetition. Top

• The slowly annealing component is due to single copy (unique) sequences. The percentages of genomes' DNAs that are unique are highly variable (see Table). Unique DNA contains DNA of structural (protein encoding) genes. Top

Interpretations

Reassociation kinetics summary for hypothetical eucaryotic nuclear DNA:

Further information

• Measurement of DNA reassociation kinetics reveals components of different kinetic complexities. When combined with microfluorometry the measurements result in a determination of repetition frequency in a genome.
• *The author has lost reference to an updated and much changed value for this component. Anyone knowing the current best estimate is asked to contact me.

This is page 112282 of Molecular Genetics by Ulrich Melcher, © 1998-2000