Properties of DNA.pptx

properties of DNA

1. Presented by D.Ragavi M.Sc.Biochemistry

2. Properties of DNA

3. Buoyant density Viscosity Hypochromisity and hyperchromicity Denaturation and Renaturation Cot curve

4. Buoyant Density of DNA A measure of the density of DNA determined by the equilibrium point reached by DNA after density gradient centrifugation. Density gradient centrifugation is a common method of separating macromolecules, particularly nucleic acids, in solution. A cell extract is mixed with a solution of CsCl to a final density of about 1.7g/cm3 and centrifuged at high speed (40,000rpm, giving relative centrifugal forces of about 200000g). The biological macromolecules in the extract will move to equilibrium positions in the CsCl gradient that reflect their buoyant densities.

5. 5. A number of factors affect the buoyant density, such as ➢ the nature of the caesium salt ➢ the presence of heavy metals or DNA-binding dyes ➢ the pH and ➢ the temperature

6. Density gradient centrifugation The density of DNA can be measured with the help of a technique known as CsCl density gradient centrifugation. A CsCl solution is set up in a centrifuge tube. The CsCl forms a concentration gradient within the tube when centrifuged at high speed, with more concentrated CsCl towards the base. As the density of the solution differs with concentration, the solution is less dense at the top and eventually gets denser toward the base. The inclusion of DNA in the solution may cause it to migrate until it reaches its density. Hence, heavy DNA will form a band in the centrifuge tube at a lower position than lighter DNA Buoyant density of majority of DNA is 1.7 g/ cm3 which is equal to density of 6 M Cscl solutions. Buoyant density of DNA changes with its “GC content”.

7. Fig. 1. Cscl density gradient centrifugation

8. Viscosity Viscosity is a measure of a fluid's resistance to flow. It describes the internal friction of a moving fluid. A fluid with large viscosity resists motion because its molecular makeup gives it a lot of internal friction. Solutions of carefully isolated, native DNA are highly viscous at pH 7.0 and room temperature (20 to 25°C). When such a solution is subjected to extremes of pH or to temperatures above 80 to 90°C, its viscosity decreases sharply, indicating that the DNA has undergone a physical change. Just as heat and extremes of pH cause denaturation of globular proteins, so too will they cause denaturation or melting of double helical DNA. This involves disruption of the hydrogen bonds between the paired bases and the hydrophobic interactions between the stacked bases. As a result, the double helix unwinds to form two single strands, completely separate from each other along the entire length, or part of the length (partial denaturation), of the molecule. No covalent bonds in the DNA are broken.

9. Hyperchromicity / Hyperchromic effect 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 addition of denaturant or by increasing the pH level. Heat denaturation of DNA, also called melting, causes the double helix structure to unwind to form single stranded DNA. When DNA in solution is heated above its melting temperature (usually more than 80 °C), the double stranded DNA unwinds to form single-stranded DNA. The bases become unstacked and can thus absorb more light. In their native state, the bases of DNA absorb light in the 260-nm wavelength region.

10. 8. When the bases become unstacked, the wavelength of maximum absorbance does not change, but the amount absorbed increases by 37%. A double stranded DNA strand dissociating to two single strands produces a sharp cooperative transition. The phenomenon of UV absorbance increasing as DNA is denatured is known as the hyperchromic shift. Hyperchromicity can be used to track the condition of DNA as temperature changes. The transition/melting temperature (Tm) is the temperature where the absorbance of UV light is 50% between the maximum and minimum, i.e. where 50% of the DNA is denatured. A ten fold increase of monovalent cation concentration increases the temperature by 16.6 °C.

11. Fig.2.The absorbance spectra of a DNA in the solution at 260nm and pH 7. Fig.3.DNA melting curve

12. Hypochromicity Hypochromicity describes a material’s decreasing ability to absorb light. Hypochromic effects The Hypochromic Effect describes the decrease in the absorbance of ultraviolet light in a double stranded DNA compared to its single stranded counterpart. Compared to a single stranded DNA, a double stranded DNA consists of stacked bases that contribute to the stability and the hypochromicity of the DNA. When a double stranded DNA is denatured, the stacked bases break apart an thus becomes less stable. It also absorbs more ultraviolet light since the bases no longer forms hydrogens bonds and therefore are free to absorb light.

13. 5. Ways to denature DNA include high temperature, addition of denaturant, and increasing the pH level. F ig.4. Hypochromic effect of DNA

14. Importance of hypochromic effect The measurement of absorption of light is important in monitoring the melting and annealing of DNA. At the melting temperature (Tm), the DNA is half denatured and half double stranded. By lowering the temperature below the Tm, the denatured DNA strands would anneal back into a double stranded DNA. When temperature is above the Tm, the DNA is denatured. Because melting occurs almost instantly at a certain temperature, monitoring the absorbance of the DNA at various temperature would indicate the melting temperature. Fig.5.

15. 5. 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. 6. This is important in creating hybrid DNAs, which consists of two DNA strands from different sources. 7. Since DNA strands can only anneal if they are similar, the creation of hybrid DNAs can indicate similarities between genomes of different organisms. Fig.6 Hypo and Hyperchromic effect of DNA

16. Denaturation and Renaturation of DNA helix Denaturation of DNA is a loss of biologic activity and is accompanied by cleavage of hydroge bonds holding the complementary sequences of nucleotides together. This results in a separation of the double helix into the two constituent polynucleotide chains. In it, the firm, helical, two-stranded native structure of DNA is converted to a flexible, single-stranded `denatured' state. Th splittin g of DNA molecule into its two strands or chains, during denaturation, is obvious becaus of the fact that the hydrogen bonds holding the bases are weaker than the bonds holding the bases to the sugar-phosphate groups. The transition from native to a denatured form is usuall ve ry abrupt and is accelerated by reagents such as urea and formamide, which enhance the aqueou e solubility of the purine and pyrimidine groups.

17. Denaturation involves the following changes : 1. Increase in absorption of ultraviolet light (= Hyperchromic effect): As a result of resonance, all of the bases in nucleic acids absorb ultraviolet light. And all nucleic acids are characterized by a maximum absorption of UV light at wavelengths near 260 nm. When the native DNA (which has base pairs stacked similar to a stack of coins) is denatured, there occurs a marked increase in optical absorbancy of UV light by pyrimidine and purine bases, an effect called hyperchromicity or hyperchromism whch is due to unstacking of the base pairs. This change reflects a decrease in hydrogen-bonding. Hyperchromicity is observed not only with DNA but with other nucleic acids and with many synthetic polynucleotides which also possess a hydrogen-bonded structure.

18. 2. Decrease in specific optical rotation : Native DNA exhibits a strong positive rotation which is highly decreased upon denaturation. This change is analogous to the change in rotation observed when the proteins are denatured. 3. Decrease in viscosity: The solutions of native DNA possess a high viscosity because of the relatively rigid double helical structure and long, rodlike character of DNA. Disruption of the hydrogen bonds causes a marked decrease in viscosity. Effect of pH on denaturation Denaturation of DNA helix also occurs at acidic and alkaline pH values at which ionic changes of the substituents on the purine and pyrimidine bases can occur. In acid solutions near pH 2 to 3, at which amino groups bind protons, the DNA helix is disrupted. Similarly, in alkaline solutions near pH 12, the enolic hydroxyl groups ionize, thus preventing the keto-amino group hydrogen bonding.

19. Effect of temperature on denaturation The DNA double helix, although stabilized by hydrogen bonding, can be denatured by heat by adding acid or alkali to ionize its bases. The unwinding of the double helix is called melting because it occurs abruptly at a certain characteristic temperature called denaturaiton temperature or melting temperature (Tm). 3. Since the G-C base pair has 3 hydrogen bonds as compared to 2 for A-T, it follows that DNAs with high concentrations of G and C might be more stable and have a higher Tm than those with high concentrations of A and T. other words, the DNA molecules containing less G-C bond denature first as G-C bond has higher thermal stability. Infact, the Tm of DNA from many species varies linearly with G-C content, rising from 77 to 100°C as the fraction of G-C pair increases from 20% to 78%. This measurement may, hence forth, be used as an index of heterogeneity of nucleic acid molecules

20. I. Nucleation reaction : In this hydrogen bonds form between two complementary single strands ; this is a bimolecular, second-order reaction. II. Zippering reaction : In this hydrogen bonds form between all the bases in the complementary strands ; this is a unimolecular, first-order reaction.

21. Cot curve History Cot analysis was first developed and utilized in the mid 1960s by Roy Britten, Eric Davidson, and associates. Cot analysis It is based upon the principles of DNA renaturation kinetics. DNA Renaturation Kinetics The rate at which heat-denatured DNA sequences in solution will renature is dependent on DNA concentration, reassociation temperature, cation concentration, and viscosity (usually not a factor if the DNA is free of contaminants). • Cot = DNA conc. (mol/L) x renaturation time in sec x a buffer factor that accounts for the effect of cations on the speed of renaturation

22. Procedure The procedure involves heating a sample of genomic DNA until it denature into the single stranded-form, and then slowly cooling it, so the strands can pair back together. While the sample is cooling, measurements are taken of how much of the DNA is  base paired  at each temperature. The amount of single and double-stranded DNA is measured by rapidly diluting the sample, which slows reassociation, and then binding the DNA to a  hydroxylapatite  column. The column is first washed with a low concentration of  sodium phosphate buffer, which elutes the single-stranded DNA, and then with high concentrations of phosphate, which elutes the double stranded DNA. The amount of DNA in these two solutions is then measured using a  spectrophotometer.

23. Analysis Since a sequence of single-stranded DNA needs to find its  complementar strand to reform a double helix, common sequences renature more rapidly than rare sequences. Indeed, the rate at which a sequence will reassociate is  proportional to the number of copies of that sequence in the DNA sample. A sample with a highly-repetitive sequence will renature rapidly, while complex sequences will renature slowly. However, instead of simply measuring the percentage of double-stranded DNA versus time, the amount of renaturation is measured relative to a C t value. The C t value is the product of C  (the initial concentration of DNA), t (time in seconds), and a constant that depends on the concentration of  cations in the buffer. Reptitive DNA will renature at low C t values, while complex and unique DNA sequences will renature at high C t values. The fast renaturation of the repetitive DNA is because of the availability of numerous complementary sequences.

24. Application to genome sequencing C t filtration is a technique that uses the principles of DNA renaturation kinetics to separate the repitative DNA sequence that dominate many eukaryotic genome from “gene-rich” single/low-copy sequences This allows DNA sequencing to concentrate on the parts of the genome that are most informative and interesting, which will speed up the discovery of new genes and make the process more efficient.