Chapter 2 Chemical and Physical Properties of DNA PDF

Chapter 2: Chemical and Physical Properties of DNA Chapter Topics I Denaturation of DNA II DNA reassociation kinetic III III. Cot curve IV Buoyant Density of DNA V Solubility 1 I. Denaturation of DNA The conversion of double he...

Chapter 2: Chemical and Physical Properties of DNA Chapter Topics I Denaturation of DNA II DNA reassociation kinetic III III. Cot curve IV Buoyant Density of DNA V Solubility 1 I. Denaturation of DNA The conversion of double helical DNA into single strands is called denaturation(Figure 2.1). The conversion of single strands back to the double-stranded structure is called renaturation. Denaturation occurs when duplex DNA molecule is subjected to Figure 2.1Denaturation and Renaturation conditions of pH, temperature, or of DNA ionic strength that disrupt hydrogen bonds.If temperature is the denaturing agent, the process is called DNA melting. Denaturation is followed by measuring the absorption of ultraviolet (UV) light at a wavelength of 260 nm (Figure 2.2). Denaturation is accompanied by an increased UV absorbance (hyperchromicity) which is Chapter 2: Chemical and Physical Properties of DNA caused by Stacking interaction of hydrophobic bases. dsDNA is hypochromic (from the Greek for ‘less colored’) relative to ssDNA. Alternatively, ssDNA may be said to be hyperchromic when compared Figure 2.2Melting of DNA with dsDNA. 2 The Tm or melting temperature is the point at which half of the double strandedDNA has separated. Factors that affect the Tm 1. G+C content The GC content (% G + C) is calculated from the fractional composition of bases as follows: The higher a DNA’s GC content, the higher its Tm. This is because G:C pairs are held by three H bonds whereas A:T pairs have only two Figure 2.3. A DNA molecule consisting of only A - T pairs will melt at approximately 69 ºC A DNA molecule consisting of Figure 2.3The dependence of melting only GC pairs will melt at temperature on relative (G + C) content in DNA. approximately 110 ºC. Chapter 2: Chemical and Physical Properties of DNA The relationship between GC content in the molecule and the melting temperature (Tm) of the molecule is as follows: %(G - C content) = 2.44(Tm - 69) 2. Ionic strength Salts stabilize the DNA double helix resulting in a higher Tm. This is because ions suppress the electrostatic repulsion between the negatively charged phosphate groups on the backbone and hence exert a stabilizing effect. 3 In pure water, DNA will melt even at room temperature. 3. pH Acids Acidic pHs result in the breakage of phosphodiester bonds between nucleotides and breakage of the N-glycosidic bond between the sugar and purine bases pH of around 4 results in the selective breakage of N-glycosidic bonds between the sugar and purines. DNA treated this way is referred to a apurinic acid, since the purines have been removed Alkali At pH a value greater than 10, extensive deprotonation (removal of a proton (H+) of the bases occurs, destroying their hydrogen bonding potential and denaturing the DNA duplexes. Alkali is preferred denaturant because, unlike acid, it does not hydrolyze the glycosidic linkages in the sugar-phosphate backbone. How to calculate the Tm 1. 2+4 rule Tm (in °C) = 2(A+T) + 4(C+G) Chapter 2: Chemical and Physical Properties of DNA (A+T): the sum of the A and T residues in the oligonucleotide, (C+G): the sum of G and C residues in the oligonucleotide. Note that the 2+4 rule is valid for DNA sequence between 14 and 18 nucleotides 2. Linear regression This method is based on the length of the DNA molecule and the GC ratio. One term, is: 4 Tm = 81.5 + 16.6(log10([SALT]/1.0 + 0.7 [SALT])) +0.41x (%GC) – 500/L, where [SALT] = [K+] + 4 [Mg+2 ]0.5 in M, (%GC) is the GC ratio, and L is the length of the sequence. II. DNA reassociation kinetics DNA reassociation kinetics is a biochemical technique that measures sequence complexity in a genome. Complexity (denoted by N) is the number of base pairs of nonrepeating DNA in a given segment of DNA. This is different from the length (L) of the sequence if some of the DNA is repeated, as illustrated in this example. For example, consider 1000 bp DNA. 500 bp is sequence a, present in a single copy. 500 bp is sequence b (100 bp) repeated 5 times: a b bbbb |___________|__|__|__|__|__| L = length = 1000 bp = a + 5b N = complexity = 600 bp = a + b Chapter 2: Chemical and Physical Properties of DNA III. Cot curve 1. The total DNA is first cleaved into fragments with an average size of about 1000 bp. 2. Fragments are heated to separate the complementary strands of each fragment. 3. Temperature is reduced to allow strand reassociation. 4. The amount of single-stranded DNA left (C/Co) is plotted against Cot. The resulting curve represents the reassociation kinetics of the DNA sample, and is known as a Cot curve (pronounced ‘cot’). 5 The equation describing reassociation kinetics: C/Co = 1/ (1 + k Cot) Where C: the concentration of single-stranded DNA at time t (moles of nucleotide/liter) ▪Co: initial concentration of single-stranded DNA ▪C/Co: fraction of single-stranded DNA ▪K: reassociation rate constant (depends on salt concentration, temperature, fragment size, viscosity, complexity of DNA, etc.) ▪t: time (seconds) In the Cot curve, Cot½ is the value at which half the component has renatured estimates the kinetic complexity of the DNA 1. Cot curve for prokaryotic DNA The Cot curve in Figure 2.4 shows same shape (simple sigmoidal shape) to different DNAs but their Cot½ differs from each other. Thus, low complexity DNA sequences reanneal faster than do high complexity sequences. Chapter 2: Chemical and Physical Properties of DNA Figure 2.3 Cot curve for prokaryotic DNA 6 2. Cot curve for eukaryotic DNA The Cot curve of DNA purified from a complex eukaryote, such as human is made up of three different components. The first component to reanneal (at a Cot½ of 10-2) is composed of highly repeated DNA sequences, with an average repetition of about 500,000 times in Figure 2.4 Cot curve for eukaryotic DNA the genome. The second component, moderately repeated DNA sequences Cot½= 1) is comprised of DNA sequences which are represented from 50 to 5,000 times in the genome. The final component, "single copy DNA" or "non-repetitive DNA" includes all the DNA which is indeed present in just one copy per genome Chapter 2: Chemical and Physical Properties of DNA but also includes many sequences which are present just a few times. Note: Repetitive DNA will renature at low Cot values, while complex and unique DNA sequences will renature at high Cot values IV. Buoyant Density of DNA Not only the melting temperature of DNA but also its density in solution is dependent on relative G:C content. 7 The density of DNA is typically slightly greater than 1.7 g/cm3, while the density of RNA is more than 1.8 g/cm3. Proteins have densities less than 1.3 g/cm3 Because of its relatively high density, DNA can be purified from cellular material by density gradient centrifugation known as isopycnic centrifugation If DNA is present in the centrifuged CsCl solution, it moves to a position where its density equivalent to that of the of CsCl solution (1.6 to 1.8 g/mL) (Figure 2.6). V. SOLUBILITY RNA is more soluble in aqueous solutions then DNA Figure 2.6 Densitygradient Ribose has a 2'-OH group where Chapter 2: Chemical and Physical Properties of DNA centrifugation deoxyribose contains a 2'-H Hydroxyl groups are polar and dissolve in water better C-H is a non-polar bond and is therefore hydrophobic RNA is less stable then DNA The hydroxyl group on the 2' carbon of ribose is more reactive than hydrogen found in deoxyribose. 8 Sample Questions on Calculating Melting temperature of DNA Q1.A 4000 kbp (kilobase-pair) DNA molecule melts at 79 ºC. What percent of the base pairs are GC pairs? Answer: 24.4 % A DNA molecule consisting of only A - T pairs will melt at approximately 69ºC, while a DNA molecule consisting of only GC pairs will melt at approximately 110ºC. The relationship between GC content in the molecule and the melting temperature (Tm) of the molecule is as follows: %(G - C content) = 2.44(Tm - 69) Substitute Tm = 79oC => %(G - C content) = 24.4 Q2. An 800bp PCR product having a GC content of 55% is synthesized in a reaction containing 50 mM KCl and 2.5 mM MgCl2. What is the amplicon’s Tm? Answer: The Molar concentration of KCL = 0.005 The Molar concentration of MgCl2 = 0.0025 Chapter 2: Chemical and Physical Properties of DNA [SALT] = 0.005 = 4 [0.0025]0.5 = 0.25 Tm = 92.3 ºC 9