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volume than a preparation of human DNA (haploid genome size 3 106 kb), and will

therefore renature far more rapidly, since there will be more molecules complemen-

tary to each other per unit volume in the case oflDNA, and therefore more chance of

two complementary strands colliding with each other. In order to compare the rates of

renaturation of different DNA samples it is usual to measureCoand the time taken for

renaturation to proceed half way to completion,t1/2, and to multiply these values

together to give aCot1/2value. The larger theCot1/2, the greater the complexity of the

DNA; hencelDNA has a far lowerCot1/2than does human DNA.

In fact, the human genome does not renature in a uniform fashion. If the extent

of renaturation is plotted against logCot(this is known as aCot curve), it is seen that

part of the DNA renatures quite rapidly, whilst the remainder is very slow to renature

(Fig. 5.8). This indicates that some sequences have a higher concentration than others;

in other words, part of the genome consists of repetitive sequences. These repetitive

sequences can be separated from the single-copy DNA by passing the renaturing

sample through a hydroxylapatite column early in the renaturation process, at a time

which gives a low value ofCot. At this stage only the rapidly renaturing sequences will

be double-stranded, and they will therefore be the only ones able to bind to the

column.

5.3.5 The nature of the genetic code

DNA encodes the primary sequence of a protein by utilising sets of three nucleotides,

termed acodonor triplet, to encode a particular amino acid. The four bases (A, C, G

and T) present in DNA allow a possible 64 triplet combinations; however, since there

are only 20 naturally occurring amino acids more than one codon may encode an

amino acid. This phenomenon is termed thedegeneracyof thegenetic code. With the

exception of a limited number of differences found in mitochondrial DNA and one or

Slowly

renaturing

DNA

Rapidly renaturing DNA

0

50

Renaturation (%)

–2

LogCot

–1 0 1 2 3 4 5

100

Fig. 5.8Cot curve of human DNA. DNA was allowed to renature at 60C after being completely dissociated

by heat. Samples were taken at intervals and passed through a hydroxylapatite column to determine the

percentage of double-stranded DNA present. This percentage was plotted against logCot(original

concentration of DNA3 time of sampling).

148 Molecular biology, bioinformatics and basic techniques