Technique for detecting repetitive DNA

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


Repetitive DNA
Technique for detecting repetitive DNA
In the year 1964, it was discovered with surprise that much of DNA in mouse cells consisted of multiple copies of same or, very similar sequences of base pairs. It is now known that in higher organisms, this repetitive DNA generally constitutes atleast 20% and can reach upto as much as more than 90% in some cases. It is believed that these repetitive sequences do not carry any genetic information and, therefore, do not form genes, but play some other structural or regulatory role. The presence of repetitive DNA can be detected by a simple method involving following procedure : The double stranded DNA is first denatured and converted into single stranded DNA by heating the DNA solution. This is accompanied with increase in optical density, a phenomenon described as hyperchromicity. The solution of single stranded DNA thus obtained is then allowed to cool slowly, so that the double stranded DNA will be formed again, which accompanies decrease in optical density (hypocromicity, Fig. 28.2). This process is called reassociation of DNA for which one should maintain fairly high concentration of DNA and a temperature 25°C below the dissociation temperature or melting temperature Tm (Tm is a temperature, at which 50% reassociation is achieved). The mixture should be allowed to undergo reassociation for a fairly long time so that sequences may come in contact with their complementary sequences (Fig. 28.3). The amount of repetitive DNA will be determined on the basis of extent of the formation of double stranded DNA in a definite period of time keeping concentration constant. The formation of double stranded DNA is actually measured over different values of a parameter which is described as Cot (conc. x time).


Hyperchromicity and hypochromicity'of DNA due to denaturation and reassociation respectively.
Fig. 28.2. Hyperchromicity and hypochromicity'of DNA due to denaturation and reassociation respectively.
 
Denaturation and reassociation of DNA containing six copies of repetitive DNA (AA;) shown by thin lines and one copy each of four different unique or single copy DNA's (B-B'; C-C’; D-D; E-E') shown by thick lines. Note that more repetitive DNA has reassociated than the single copy DNA. This principle is used for determining the proportion of repetitive DNA in a genome (redrawn from Sci. Amer. 222: April, 1970).
Fig. 28.3. Denaturation and reassociation of DNA containing six copies of repetitive DNA (AA;) shown by thin lines and one copy each of four different unique or single copy DNA's (B-B'; C-C’; D-D; E-E') shown by thick lines. Note that more repetitive DNA has reassociated than the single copy DNA. This principle is used for determining the proportion of repetitive DNA in a genome (redrawn from Sci. Amer. 222: April, 1970).

If solutions of different concentrations are to be compared, same Cot value can be achieved by altering the time allowed for reassociation. The double stranded DNA formed at a definite Cot value can be estimated by measuring the optical density of solution in a spectrophotometer, because the density of a solution goes down as the proportion of double stranded DNA increases. The proportion of double stranded DNA formed at a definite Cot value can also be estimated by separating double stranded DNA thus formed from single stranded DNA. This can be achieved, if solution is allowed to pass through a column of hydroxyapatite crystals, which have the capacity of retaining double stranded DNA and permitting single stranded DNA to pass through (Fig. 28.4).
 
Technique used for measuring the extent of reassociation by separating reassociated double stranded DNA from remaining single stranded DNA. Mixture of double stranded and single stranded DNAs is passed through a column of hydroxyapatite crystals, which retains the double stranded DNA, and allows single stranded DNA to pass through. Double stranded DNA can be eluted by raising temperature and its quantity measured (redrawn from Sci. Amer. 222, April, 1970).
Fig. 28.4. Technique used for measuring the extent of reassociation by separating reassociated double stranded DNA from remaining single stranded DNA. Mixture of double stranded and single stranded DNAs is passed through a column of hydroxyapatite crystals, which retains the double stranded DNA, and allows single stranded DNA to pass through. Double stranded DNA can be eluted by raising temperature and its quantity measured (redrawn from Sci. Amer. 222, April, 1970).

Reassociation patterns of double stranded DNA from various sources. All curves are typical S-shaped indicating homogeneity, but they are displaced to different values to Cot due to genome size.
Fig. 28.5. Reassociation patterns of double stranded DNA from various sources. All curves are typical S-shaped indicating homogeneity, but they are displaced to different values to Cot due to genome size.


If a DNA sample has high proportion of repetitive DNA, reassociation will be faster, with higher degree of reassociation achieved at lower Cot values. Reassociation rate will also depend on genome size, because in a small genome, higher degree of repetition will be available at the same DNA concentration. Therefore, bacterial DNA with small genome size will reassociate faster, even though no repetitive DNA is found there, so that the range of Cot values for complete reassociation is small. In eukaryotes with a bigger genome size, on the other hand, the range of Cot values is large suggesting a longer time required for reassociation. But a specific fraction of DNA reassociates faster, indicating the presence of repetitive DNA. The patterns of reassociation in a number of organisms are shown in Cot curves in Figures 28.5 and 28.6. During the last more than 20 years, hundreds of plants and animals have been analysed for the relative proportion of repetitive DNA in their genomes (Table 28.2). In our own country, Dr. P.K. Ranjekar at National Chemical Laboratory, Poona has been involved in this kind of work.
 
Reassociation patterns of bacterial and calf DNA. Note that the reaction on calf DNA takes place in two stages, one early representing repetitive DNA and the other representing single copy DNA. Midpoints of these two stages are indicated by dotted vertical lines, separated by a factor of 1000,000 (for details see text), (redrawn from Sci. Amer. 222:. April, 1970).
Fig. 28.6. Reassociation patterns of bacterial and calf DNA. Note that the reaction on calf DNA takes place in two stages, one early representing repetitive DNA and the other representing single copy DNA. Midpoints of these two stages are indicated by dotted vertical lines, separated by a factor of 1000,000 (for details see text), (redrawn from Sci. Amer. 222:. April, 1970).

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