DNA Denaturation and Renaturation, Cot curves

DNA Denaturation and Renaturation, Cot curves

1. DNA Denaturation and Renaturation , Cot curves

2. Absorption of UV light Nucleic acids exhibit characteristic absorption in the ultraviolet region. This absorption is due to the conjugated double bonds and ring system of constituent purine and pyrimidines. The more ordered the structure, the less light is absorbed. Therefore , free nucleotides absorb more light than a single-stranded polymer of DNA or RNA and these in turn absorb more light than a double-stranded DNA molecule. The maximum absorption is at 260 nm (A 260 ) and the minimum absorption is at 230 nm. Absorption is proportional to the concentration of the molecule, with a value of 0.02 units per μg DNA per ml. For example, three solutions of double-stranded DNA, single-stranded DNA and free bases each at 50 μg /ml have the following A 260 values: Double-stranded DNA A 260 = 1.00 Single- stranded DNA A 260 = 1.37 Free bases A 260 = 1.60 Therefore, double stranded DNA is said to hypochromic and the bases are said to be hyperchromic .

3. Denaturation of DNA Molecules The ordered state of DNA helix, which is, originally present in nature is called the native form . The two strands of DNA readily come apart when the hydrogen bonds between its paired bases are disrupted. This can be accomplished by heating a solution of DNA or by adding acid or alkali to ionize its bases. This unwinding of DNA double helix is called melting and a transition from the native to the denatured state is called denaturation. Denaturation of DNA molecule can be studied by measuring its absorbance at a wavelength of 260 nm. As the DNA is subjected to an increase in temperature, A 260 starts increasing because of DNA. When both the strands are completely separated at a particular higher temperature, there is maximum A 260 that indicates complete denaturation of the molecule has taken place. The temperature at which half of the helical structure of DNA molecule is lost is called its melting temperature (Tm). A convenient parameter to analyze melting transition. Molecules rich in GC pairs have a higher Tm than those having abundance of AT base pairs because GC base pairs are more stable and held together by three hydrogen bonds . Such DNA molecules require more energy and hence temperature to denature.

4. Denaturation involves changes Denaturation converts the firm, helical two-stranded native structure of DNA to a flexible, single-stranded denatured state. The splitting of DNA molecule into its two strands or chains is obvious because of the fact that the hydrogen bonds are weaker than the bonds holding the bases to the sugar phosphate groups. Denaturation involves following changes : Increase in absorption of ultraviolet light: Due to 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 wavelength near 260nm. When the native DNA (which has base pairs similar to a stack of coins) is denatured, there occurs a marked increase in optical absorbance of UV light by pyrimidine and purine bases, an effect called hyperchromicity or hyperchromism which is due to unstacking of the base pairs. This change reflects a decrease in hydrogen bonding. Decrease in specific optical rotation: Native DNA exhibits a strong positive rotation which is highly decreased upon denaturation. (same as in proteins) 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

5. For example (the absorption of ultraviolet light ), if a solution of double-stranded DNA has a value of A 260 =1.00, a solution of single-stranded DNA at the same concentration has a value of A 260 =1.37. This relation is often described by stating that a solution of double-stranded DNA becomes hyperchromic when heated. The following features of this curve should be noted: The A 260 remains constant up to temperatures well above those encountered by most living cells in nature. The rise in A 260 occurs over a relatively narrow range of 6-8 ℃ The maximum A 260 is about 37% higher than the starting value Please note that during melting all covalent bonds, including phosphodiester bonds, remain intact. Only hydrogen bonds and stacking interactions are disrupted . Denaturation and absorbance

6. Compounds like urea and formamide /formaldehyde are capable of hydrogen bonding with the DNA bases. Hence, they maintain the unpaired state of DNA molecules and result in lowered Tm value, upon melting. Formaldehyde reacts with NH 2 groups DNA bases and eliminates their ability to hydrogen bond. Hence addition of formaldehyde causes a slow and irreversible denaturation of DNA. There is always a fluctuation in the structure of DNA. The double-stranded regions frequently open to become single-stranded bubbles. This phenomenon is called breathing, which enables specialized proteins to interact with DNA molecule and to read its encoded information. Breathing occurs more often in regions rich in AT pairs than in regions rich in GC pairs. There are many proteins that can unwind a DNA helix. An example of this type of protein is gene 32 of E. coli phage T4 , commonly called the 32-protein . This protein binds tightly to the bases of single-stranded DNA. The individual molecules of the 32-protein prefer to line up adjacent to one another along a single strand. Binding of the first molecule is made possible by the breathing of the DNA. Denaturation of DNA can also be accomplished by treatment with alkali. Since DNA is quite resistant to alkali hydrolysis, this procedure is the method of choice for denaturing DNA, because heat treatment may often break the phosphodiester bonds and may result in yielding broken fragments of DNA. How can we achieve Denaturation

7. DNA Renaturation Denatured DNA will renature to re-form the duplex structure if the denaturing conditions are removed (that is, if the solution is cooled, the pH is returned to neutrality, or the denaturants are diluted out). Renaturation requires re-association of the DNA strands into a double helix, a process termed reannealing . For this to occur: (1) Strands must realign themselves so that their complementary bases are once again in register (NUCLEATION PROCESS) (2) Helix can be zippered up (Figure 12.19 ). Renaturation is dependent on DNA concentration and time . Many of the realignments are imperfect, and thus the strands must dissociate again to allow for proper pairings to be formed. The process occurs more quickly if the temperature is warm enough to promote diffusion of the large DNA molecules but not so warm as to cause melting.

8. Renaturation Rate and DNA Sequence Complexity— C t Curves The renaturation rate of DNA is an excellent indicator of the sequence complexity and the size of the DNA . For example, bacteriophage T4 DNA contains about 2x10 5 nucleotide pairs, whereas Escherichia coli DNA possesses 4.64x10 6 . E. coli DNA is considerably more complex in that it encodes more information. Or we may say that for any given amount of DNA (in grams), the sequences represented in an E. coli sample are more heterogeneous, that is, more dissimilar from one another, than those in an equal weight of phage T4 DNA. Therefore , it will take the E. coli DNA strands longer to find their complementary partners and reanneal . This situation can be analyzed quantitatively .

9. If c is the concentration of single-stranded DNA at time t, then the second-order rate equation for two complementary strands coming together is given by the rate of decrease in c: -- dc / dt = k 2 c 2 where k 2 is the second-order rate constant . Starting with a concentration, C , of completely denatured DNA at t 0, the amount of single-stranded DNA remaining at some time t is C / C 0 = 1/( 1 + k 2 C t ) where the units of C are moles of ntd per L and t is in seconds. Then the time for half of the DNA to renature (when C / C = 0.5), according to the second order rate equation, is defined as t = t 1/2 . Then, 0.5 = 1/( 1 + k 2 C t 1/2 ) and thus 1 + k 2 C t 1/2 comes out to be 2 yielding C t 1/2 = 1/ k 2 A graph of the fraction of single-stranded DNA reannealed ( C / C ) as a function of C t on a semilogarithmic plot is referred to as a C t (pronounced “cot ”) curve ( Figure). DNA Sequence Complexity and C t Curves

10. C t Curves