Reassociation kinetics

K. Ritz, B. S. Griffiths, V. L. Torsvik, and N. B. Hendriksen, Analysis of soil and bacterioplankton community DNA by melting profiles and reassociation kinetics, FEMS Microbiol. Lett. 749 151 (1997). 

For theTrematoda and Cestoda, both reassociation kinetics/Cot analysis and direct measurement of the DNA content of Feulgen-stained nuclei have been used to estimate genome size. As relatively few taxa have been examined, it is difficult to draw any firm conclusions although the available data suggest much less extreme variation than in the Turbellaria and the absence of step-like intrageneric variation (Table 2.3). 

Studies of overall genome composition based on reassociation kinetics (Simpson et ai, 1982 Cox et ai, 1990 Marx et a/., 2000) and analysis of fully sequenced bacterial artificial chromosome (BAC) clones from the 5. mansoni genome project show that platyhelminth genomes contain abundant highly and moderately repetitive sequence (Fig. 2.1). Much of the repetitive DNA comprises two classes of integrated mobile elements class I elements, which include long terminal repeat (LTR) retrotransposons and retroviruses, non-LTR retro-transposons and short interspersed nuclear elements (SINES) and transpose via an RNA intermediate, and class II elements (trans-posons), which transpose as DNA (Brindley et ai, 2003). Additionally, small dispersed or tandemly repeated sequences are common. A wide variety of these sequences have been isolated and characterized from a variety of taxa (Table 2.4). 

Schafer, A. Neuman, K. H. The influence of gibberellic acid on reassociation kinetics of DNA of Daucus carota L. Planta, 1978, 143, 1-4. 

In addition to the exclusionary value, it is necessary to know the percent G + C content to maximize DNA reassociation kinetics. Most faithful pairing of complementary nucleotide sequences occurs at Tm — 25°, that is, a temperature 25° below the midpoint of the thermal melt... 

The degree of repetitiveness in the segments of total mouse DNA is determined by measuring C0t values for several fractions of the genome. The dotted line indicates estimated values. The reassociation kinetics of eukaryotic genomes typically reveal three primary classes of sequences repetitive sequences that reanneal quickly (a), sequences of intermediate complexity (b), and unique sequences that reanneal slowly (c). 

Reassociation kinetics. (A) The reassociation of denatured DNA, initial concentration c0, is followed as a function of time t on a linear scale. (B) The same data is shown on a logarithmic scale. 

Figure 3.6. Reassociation kinetics of mouse, human and chicken DNAs and their major components. Results obtained with E.coU DNA (open circles top left frame) are shown for the sake of comparison. The solid lines through the experimental points (solid circles) are the overall profiles resulting from the analysis of kinetic classes (broken lines). Cgt is the product of initial DNA concentration by reassociation time. (From Soriano et al., 1981). Chicken data are from Olofsson and Bemardi (1983).

Soriano P., Macaya G., Bernardi G. (1981). The major components of the mouse and human genomes reassociation kinetics. Eur. J. Biochem. 115 235-239. 

Steinert M, Van Assel S. Sequence heterogeneity in kinetoplast DNA Reassociation kinetics. Plasmid 1980 3 7-17. 

Measure by spectrophotometer the DNA in the 0.12M PB and 0.48Af PB fractions. The single-copy and single-stranded DNA should be in the 0.12M PB fractions. For Drosophila an incubation to C t 200 usually is enough (50% of the input DNA is in the 0,11M PB fractions). Sometimes the spectrophotometer readings are not accurate (the phosphate buffer and/or the HAP may alter readings) you may wish to add some labelled total DNA from the same DNA preparation to follow the reassociation kinetics by scintillation counting. 

Fig. 1. Reassociation kinetics of nucleic acids isolated from various sources. Incubation reactions were usually carried out in 0.12 M phosphate buffer at 60°C before analysis on hydroxyapatite. Total mouse DNA, 2 mg/ml, was mixed with H-labeled single copy DNA, 1 ju.g/ml, previously fractionated at Cat 220 and E. coli [ C]DNA, 10 /ng/ml, before reassodation to the Cat values shown. The MS-2, poly(U) - - poly(A) and T< reassociation profiles are from Britten and Kohne (1968). The AT-rich satellite DNA was isolated from total mouse DNA by CsCl gradient centrifugation before reassociation as described previously (Church, 1973). All DNA s were sheared to a single-stranded molecular weight of approximately 120,000 before being heat denatured for 10 minutes at 100°C in 0.03 M phosphate buffer.

Britten s group (18,19) observed that many vertebrate DNA s, especially if sheared to smaller pieces, will reassociate considerably faster than one would expect. This paradox, discovered in 1966, gave rise to the hypothesis that certain relatively short sequences of bases were repeated hundreds of thousands of times. It was also shown that the extent of reassociation of DNA was a measure of the evolutionary state of the species. Furthermore, heteroassociation (i.e., between DNA strands derived from different species) was shown to be a measure of the evolutionary relation between species. Finally, most eucaryotic DNA s contained one (or several) satellite components, which formed up to 10% of total DNA and showed very fast reassociation kinetics. 

Renaturation of DNA is governed by reassociation kinetics. The reassociation rate obeys the Law of Mass Action (Section 2.3) and is concentration dependent, i.e. the higher the concentration of DNA, the faster the renaturation. The relationship between the initial DNA concentration (Q) and the concentration of unrenatured DNA (C) at time (/) is given by the equation ... 

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