Kinetic classes of DNA and denaturation kinetics
Answers
Explanation:
Kinetic Classes of Genomic DNA
Learning Objectives
Chapter 4 of The Cell: A Molecular Approach provides good background for this material.
Be able to work with the main equations for reassociation kinetics, and understand the units and terms for these equations.
Be able to interpret DNA melting curves and C0t curves.
Be able to explain the concept of sequence complexity X
Know the characteristics of the three kinetic classes of genome sequences, and know which types of sequences are found in each class.
Up to this point, we have been studying eukaryotic genomes from a very structural and mechanistic perspective. We have largely ignored the whole purpose for its existence, that being the storage and expression, and replication of genetic information. For the next four readings, we will focus on the information contained in the genome, and its organization within the chromosomes.
DNA denatures, or melts, according to bonding strength between strands
Purine and pyrimidine bases have a peak absorbance at 260 nm. For ssDNA, the absorbance of the DNA molecule is about the same as would be predicted by adding up the absorbances of each individual nucleotide. In duplexed DNA, the total A260 is less than the sum of the absorbances of the constituent nucleotides, due to electronic interactions between the stacked bases. This is referred to as the hypochromic effect. As the DNA melts (or denatures), the amount of single-stranded DNA increases, relative to the amount of double-stranded DNA. Consequently, the absorbance also increases.
The sequences with more AT base pairs are easier to denature and start to increase in absorbance first. The GC-rich sequences take longer to denature and rise in absorbance.
When melting DNA in experimental conditions, a quantity called TM is observed. TM is the temperature at which half of the DNA is denatured, or has become single-stranded. Since TM is directly related to base composition, it can be used to estimate the composition of an unknown DNA sequence.
The relationship between TM and percentage of GC base pairs is a positive linear relationship with a very strong correlation.
The time taken for DNA strands to reassociate increases as a function of genome complexity
Just as DNA melting can tell us something about the sequences present, so can reassociation. The reassociation of DNA molecules in solution is described by the equation:
Where:
C is the concentration of ssDNA in mol/L
dC is change in concentration of ssDNA with respect to time
t is time in seconds
k is a second order rate constant (L mole-1 sec-1)
The equation can be rearranged to a more convenient form:
Now, in this equation, C0 is the initial concentration of ssDNA at time zero. From this, we can find the value of C0t1/2.This is the value of C0t at which annealing has proceeded to half completion (C/C0=0.5).
We can also state this relationship:
Complexity
Complexity (X) is defined as the longest non-repetitive sequence that can be derived from a sequence. The complexity of a population of uniformly sized DNA molecules can be measured with the following equation:
X=kC0t½
where k has been determined under standard conditions (0.18M cations [eg. Na+], 400 nucleotide fragment size) as approximately 5 x 105 L bp mole-1 sec-1. Rearranging the equation, we see that:
C0t½=X/k
Therefore, C0t½ increases with the complexity of the DNA. We can use this relation to measure genome size as shown in the figure at right.
Redrawn from Russel, P.J. (1986) Genetics Fig. 7-25a. C0t plots showing the renaturation of DNA from organisms with small genomes: the bacterium E.coli, the bacterial viruses T2 and Lambda, and the animal virus SV40.
In this article we will discuss about the repeated sequence of chromosomal DNA.
Eukaryotic genomes contain large amount of repetitive sequences, sometimes present in hundreds or thousands of copies per genome. The understanding of repetitive sequences is based on studies conducted on denaturation (separation of DNA double helix into its two component strands) and renaturation (re-association of the single strands into stable double-stranded DNA molecules) of DNA.
The two strands of a DNA molecule are held together by weak non-covalent bonds. When DNA is warmed in saline solution, a temperature is reached when two strands begin to separate, leading to single-stranded molecules in solution. This is called thermal denaturation or DNA melting.
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The progression of thermal denaturation can be followed by observing increase in absorbance of the dissolved DNA. The nitrogenous bases of DNA absorb ultraviolet radiation with an absorbance maximum near 260 nm. In single stranded DNA, the hydrophobic interactions caused by base stacking are increased which increases the ability of the bases to absorb ultraviolet radiation.
The temperature at which the shift in absorbance is half completed is called the melting temperature (Tm) of DNA. The higher the GC content of the DNA, the higher the Tm. The reason being that there are 3 hydrogen bonds between G and C which confer stability on GC pairs, in comparison with AT pairs that are joined by two hydrogen bonds. Thus AT rich sections of DNA melt before the GC rich.
When denatured DNA is cooled slowly, the single strands reassociate to form double-stranded molecules, and properties of double helical DNA are restored, that is, it absorbs less ultraviolet light. This is called renaturation or reannealing. As described later, the property of reannealing has led to the development of methodology called nucleic acid hybridisation.
Britten and Kohne (1967) studied renaturation kinetics of DNA and discovered repeated sequences.
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Walker (1969) distinguished 3 kinetic classes of DNA:
Fast reannealing fraction or highly repetitious DNA,
Intermediate reannealing fraction or moderately repetitious DNA, and
The slow annealing unique or single copy fraction.
Kinetic Classes of DNA:
1. Highly Repeated DNA Sequences:
Also called reiterated or redundant DNA. Consists of sequences present in at least a million copies per genome, constitutes about 10% of the total DNA in vertebrates. Such sequences are usually short, about a few hundred nucleotides long, and present in clusters in which the given sequence is repeated over and over again without interruption in tandem arrays (end-to-end manner). Highly repeated sequences include the satellite DNAs, minisatellite DNAs and the microsatellite DNAs.
Satellite DNA:
Consists of short sequences about 5 to 100 bp in length. During density gradient centrifugation, satellite DNA separates into a distinct band, because the base composition of satellite DNA is different from that of bulk DNA. A species may have more than one satellite sequence as in Drosophila virilis which has 3 satellite sequences, each 7 nucleotides long.
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Satellite DNA is present around centromeres in centromeric heterochromatin. In humans, 3 blocks of satellite DNA are present in the secondary constrictions of chromosomes 1, 9 and 16. A fourth block is present at the distal portion of the long arm of the Y chromosome.
Minisatellite DNA:
These usually occur in clusters with about 3000 repeats, their size ranging from 12 to 100 bp in length. Minisatellite sequences occupy shorter stretches of the genome than the satellite sequences. Minisatellites are often unstable and the number of copies of minisatellites can increase or decrease from one generation to the next. The length of the minisatellite locus could vary within the same family, and in the population (polymorphism). Changes in minisatellite sequences can affect expression of nearby genes.