ASSIGMENT

ON

HYPERCHROMICITY

MEANING OF HYPERCHROMICITY - Hyperchromicity is the increase of absorbance (optical density) of a material. The most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is denatured. The UV absorption is increased when the two single DNA strands are being separated, either by heat or by of a chemical.

DNA's hyperchromic effect - that ss DNA absorbs more UV than does ds DNA.

Example of hyperchromic effect - An insect, which can see in the UV range, would see the hyperchromic effect something like that shown to the below. At first the DNA solution is only a little violet. If it is boiled and then slowly cooled, it ends up a little more violet than it started, but if it is rapidly cooled it becomes most violet. The reason that this happens is that in ds DNA the pi-electrons in the aromatic rings are more o nstrained because the H-bonded rings are in sandwich layers - overlapping with each other. But if the H-bonds are "boiled" away, the sandwich no longer exists and the pi-electrons are more free to move into different energy levels and thus able to absorb more UV energy.

Stability of the Double-Helix depend upon the following interactions :

1) Hydrophobic Interactions: The ribose-phosphate is hydrophilic on the outside of the chain. The bases are hydrophobic on the inside.

2) Stacking Interactions: Van der Waals forces Relatively weak but additive Caused by the planar nature of the bases.

3) Hydrogen Bonding: Actually not the strongest of interactions. Facilitates stacking.

4) Electrostatic Interactions: Both within chains and between chains. Phosphate groups try to get as far away from each other as possible.

There are two basic approaches to denaturing double-stranded DNA :

1) chemical treatment.2) Heating

chemical denaturants can be divided into three classes :

a) pH: Bases like NaOH raise the pH until the H+ shared between the N-base electronegative centers ( N-H and O= ) is stripped from the H-bond. Loss of H-bonds between two complementary strands results in strand separation.

b) Competitive Denaturants : Compounds like urea and formaldehyde contain functional groups that can form H-bonds with the electronegative centers of the N-bases. At high concentrations (8M urea or 70% formamide) of the denaturant, the competition for H-bonds favors interactions between the denaturant and the N-bases rather than between complementary bases. As a result, the two strands separate.

c) Covalent Modification Denaturants : Reactive aldehydes like formaldehyde and glyoxal can covalently modify the electronegative centers of the N-bases and thereby block the formation of H-bonds between complementary bases.Covalent modification is reversible.

1) Heat Denaturation : Consider what happens when we heat a nucleic acid solution - say the E. coli genome. To prepare the DNA for this experiment, we shear it up into small pieces (approximately 500 bp long) and heat it slowly while monitoring the A260.

 

As the temperature rises, the kinetic motion of molecules in solution increases.The initial A260 is stable until, over an interval of approximately 5 degrees C, the A260 suddenly increases by approximately 40%.This increase in absorbance is referred to as theHyperchromic Shift.

REASON OF HYPERCHROMIC SHIFT :

The hyperchromic shift is due to the melting of the double helix into two single strands. The increased rotational freedom of the N-bases on strand separation accounts for the observed increase in absorbance.

DENATURATION OF DNA :

If we heat up a tube of DNA dissolved in water, the energy of the heat can pull the two strands of DNA apart (there's a critical temperature called the Tm at which this happens). This process is called 'denaturation'; when we've 'denatured' the DNA, we have heated it to separate the strands.

Denaturation rate (i.e. the amount of denaturant required to do the DNA) depends on adenine solubility -- not on the relative number of double and triple hydrogen bonds.The more soluble adenine is in the denaturant, the less reagent required to denature.AT-Rich region melt first because they have only two bonds. Heat, pH, and temperature extremes will also destabilize and thereby denature DNA.

DNA melting temperature ( Tm) :

For molecular biology applications such as PCR, sequencing or microarrays, it is important to determine the melting temperature of DNA, or Tm. The Tm is defined as the temperature in degress Celsius, at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands.

Note that ‘melting’ in this sense is not a change of aggregate state, but simply the dissociation of the two molecules of the DNA double helix.

If a homogenous solution of identical double-stranded DNA molecules is heated, the strands dissociate increasingly at higher temperatures:

In the example above, the Tm would be 60°C.

The main factors which affect the melting temperature -

a) The GC content of the nucleic acid sample- This is due to the fact that AT base pairs share 2 H-bonds while GC base pairs share 3 H-bonds.

b) SALT - Tm is sensitive to Na+ concentration.Na+ acts to shield the negative charges of the sugar-phosophate backbone from interacting with one another. The repulsion between the negatively charged phosphate backbones is the major force destabilizing the double helix, therefore increasing Na+ concentration increases helix stability and decreasing Na+ concentration decreases helix stability.

c) DNA hybrid length - The longer the DNA hybrid is, the more H-bonds there are holding the two strands together. The longer the hybrid, the more H-bonds that must be simultaneously broken for the two strands to separate. This is known as the 'zipper effect' after the (in) famous Canadian inventor Zippy. For our purposes we will only consider the two extremes of the zipper effect. For this course we will only consider the extemes of hybrid length - hybrids less than 50 bp (short) and those around 500 bp (long) in length.

D) The most complex factor is the sequence of the DNA. The sequence has an impact on the Tm for a number of reasons:

• The nucleotide pair ‘A-T’ has a weaker bond than the nucleotide pair ‘G-C’.

• Nucleotides on the same strand can interact with each other, forming so-called secondary structures such as internal loops; these

structures compete with the formation of the double helix and can thus increase the Tm.

Neighbouring nucleotide pairs can interact with each other. It is energetically favourable for nucleotide pairs to be neighboured with other nucleotide pairs. This so-called stacking effect decreases the Tm.

Importance of hyperchromic effect -

The melting temperature (Tm), occurs almost instantly at a certain temperature, monitoring the absorbance of the DNA at various temperature would indicate the melting temperature. By being able to find the temperature at which DNA melted and annealed, scientists are able to separate DNA strands and anneal them with other DNA strands. This is important in creating hybrid DNAs, which consists of two DNA strands from different sources. Since DNA strands can only anneal if they are similar, the creation of hybrid DNAs can indicate similarities between genomes of different organisms.

Tm is a measure of hybrid stability - The quantitative analyses of the melting behaviour of many naturally occuring DNAs is summarized in the formula (note: this formula describes the Tm of long DNA hybrids)

Tm = 81.5 oC + 0.41 ( % GC ) + 16.6 log [Na+ ]

note that the % GC term only holds over a limited range (45 to 75%) as does the salt term ( 0.01 to 0.4 M ). note that the salt term is negative in this range.

For short DNA hybrids, the 'zipper effect' alters the formula to

  Tm = 2 oC (A+T) + 4 oC (G + C)

 Renaturation of DNA - The reannealing of two single strands is a bi-molecular reaction. Two complementary single strands must meet one anotherand then complementary base pairs form between the two strands. Because this is a bi-molecular reaction, the rate of the reaction depends on the concentration of the reactants - the two complementary strands.

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Renaturation of DNA The following example for cot analysis -

Reannealing reaction conditions are therefore chosen to maximize the rate at which hybrids form. The curve below shows how the rate of reannealing depends on temperature. To maximize the rate of reannealing, we typically hybridize 15 oC below the Tm of the hybrid. Hybridization is also done at high salt to minimize the repulsion of the sugar-phosphate backbones. Hybridization conditions are always set to maximize the rate of hybrid formation. The rate at which hybridization occurs is therefore a direct measurement of the concentration of the complementary strands in solution. In order to visualize this concept, consider the reannealing of a variety of DNA samples.

To standardize conditions : Each DNA sample is sheared to random fragments 500 bp long.Each DNA sample is at the same DNA (50 ug/ml) and NaCl concentration(1M).Each sample is heated to boiling to denature it and then held at (Tm-15 oC) while the amount of DNA remaining single stranded is monitored.

The % single stranded is graphed as a function of

Cot - the initial DNA concentration (Co) times time (t)

Since the reaction follows bimolecular kinetics, we can obtain a value

Cot1/2 = Cot at which 1/2 of the DNA has reannealed

Cot1/2 measures the rate of reannealing which is inversely proportional to the concentration of complementary sequences being examined.

Small Cot1/2 values indicate that the complementary sequences are at high concentration.(they reanneal very quickly, t is small)Large Cot1/2 values indicate that the complementary sequences are at low concentration.(they reanneal very slowly, it is large)

The lambda genome consists of 50,000 bp of unique sequence.The DNA reanneals as a single kinetic component (a single sigmoidal curve). The midpoint of the curve provides the value for Cot1/2 - the time required for half of the single

strands to be in double stranded form at a defined initial DNA concentration.

In contrast to the above example, the E. coli genome consists of 5,000,000 bp of unique sequence. Again, plotting the % single stranded as a function of Cot, we observe a single sigmoidal curve confirming that the E coli genome reanneals as a single kinetic class.

Notice that the Cot curve has shifted to the right relative to the lambda Cot curve. The increase in Cot1/2 reflects the number of copies of the E coli genome in solution as compared to the lambda genome. Since the E coli genome is 2 orders of magnitude larger than the lambda genome, any given 500 bp fragment will be present at 100 x lower concentration in the E coli genome sample.

This can be illustrated by considering what happens when the DNA sample consists of equal mass amounts of the lambda and E coli genomes (each at 25 ug/ml)

The Cot curve now has two kinetic components,each comprising about 1/2 of the total DNA in the sample :a) one with a Cot1/2 of the lambda genome,b) one with a Cot1/2 of the E coli genome.

IN CASE OF EUKARYOTES :

Unlike most prokaryotes, the Cot curves of eukaryotic genomes are complex curves. Modeling based on bimolecular kinetics allows us to 'fit' the data to define three kinetic classes that differ in their repetition frequency in the genome.

a) The first class represents a small portion of the genome (typically 10% or so) but is very highly repeated (10,000s of copies). These are short highly clustered repeated sequences found at eukaryotic telomeres and centromeres. Dispersed copies of these simple sequence repeats are also common.

b) The second class is called moderately repetitive. This class contains longer sequences which are repeated 100s to 1000s of times in the genome. Ribosomal RNA genes, histone genes and a few others fit into this class. Another major component are the transposable elements - a class of sequence which has the capacity to replicate and move to new positions in the genome. The portion of the genome in this portion of the Cot curve varies widely.

c) The third kinetic class is the unique sequence of the genome. In this class we find most the protein coding sequences of the genome. This class typically contains the majority of the sequence in the eukaryotic genome.