Chemical complexity vs sequence (kinetic) complexity

Content
Organization of Genetic Material 2.  Repetitive and Unique DNA Sequences
Chromosomal DNA Content and C-value paradox
Repetitive DNA 
Technique for detecting repetitive DNA
Chemical complexity vs sequence (kinetic) complexity
Repetitive DNA in the form of satellite DNA
Squash dot hybridization
Selfish DNA
Chemical complexity vs sequence (kinetic) complexity
Renaturation of DNA in solution as discussed above, depends on random collisions between the complementary strands, which follows second order kinetics, since concentrations of both (not only one of them) the complementary strands will influence the rate of reaction. The rate of reaction, when expressed through differential calculus is given by


(C = concentration of single stranded DNA at time t)
(K = reassociation rate constt.)

By integrating the above equation (consult a book on Integral Calculus, if necessary), between the limits of C0 at t = 0 and C at time t1/2 (when reaction is half complete), we get the following expression.

In view of the above, reassociation of any particular DNA can be described by the rate constant K (in units, nucleotide moles liter-1 sec-1) or in the form of its reciprocal i.e. Cot1/2 (in nucleotides moles x sec/liter). It is obvious that reassociation depends on concentration (Co) and time of incubation (t), i.e. Cot as described above. A higher Cot1/2 means slower reaction and lower Cot1/2 meant faster reaction. This parameter Cot1/2 varies for different organisms and also for different DNA fractions from the same organism.

The Cot1/2 of a reaction will be directly proportional to the DNA content, if there is no repetition of sequences within a single genome. For instance, if the bacterial genome has 0.004 pg DNA (4.2 x 106 bp) and if Co is 12 pg per unit volume, it will have 3000 copies of each DNA sequence. Contrary to this if a eukaryotic genome is 4 pg, then there will be only 3 copies in 12 pg i.e. concentration is 1000 times less than in bacterial genome, provided there is no repetition of sequences in the genome of 4 pg. In view of this, Cot1/2 will indicate the length of all the different sequences (represented only once) in a genome, which will be less than the length of total DNA in a genome when there is repetition. This will be described as kinetic complexity of the genome, and can be determined by knowing Cot1/2 and comparing it with Cot1/2 of a genome with known complexity (e.g. E. coli, which has a genome = .004 pg DNA = 4.2 x 106 bp with Cot1/2 = 9). The following relationship is used for calculating the kinetic complexity.


Figure 28.5 shows reassociation curves of several DNAs, which are all kinetically pure, meaning that DNA is homogeneous from the point of view of complexity. In eukaryotes, the genome contains more than one such pure components as shown in the comparison made in Figure 28.6. In this figure calf DNA has been shown to have two components, each with its characteristic Cot1/2 value.

However, more than two such components can also be found as shown for wheat in Figure 28.7. Proportion of each component is determined by using the formula 1 - C/C0 (C = concentration at t1/2 for the corresponding component). From this proportion, chemical complexity of the component can be determined, if chemical complexity of the genome is known. For instance, if genome size is 12 x 108 bp and the component represents 25% of the genome, then the chemical complexity of this component is 3 x 108 bp. If kinetic complexity is known from the equation given earlier , then repetition frequency (f) of repetitive DNA component can be determined using the following formula :



The results of an exercise to determine the size of three different DNA components and their Cot1/2 values along with kinetic complexity and the repetition frequencies are shown in Figure 28.8.

Reassociation pattern of wheat genomic DNA showing more then two homogeneous components (fast, intermediate and slow reassociation)
Fig. 28.7. Reassociation pattern of wheat genomic DNA showing more then two homogeneous components (fast, intermediate and slow reassociation)
 
Reassociation kinetics of eukaryotic DNA, showing calculation of complexity and repetition frequency.
Fig. 28.8. Reassociation kinetics of eukaryotic DNA, showing calculation of complexity and repetition frequency.