Physiochemical properties of dna

Discription about physiochemical properties of dna.

1. Physiochemical properties of dna KESSIYA T PETER BSC MICROBIOLOGY INDIRA GANDHI ARTS AND SCIENCE COLLEGE ,NELLIKUZHI

2. PHYSIOCHEMICAL PROPERTIES OF DNA ABSORPTION OF LIGHT BY DNA BUOYANT DENSITY OF DNA DENATURATION OF DNA RENATURATION OF DNA SOLUBILITY OF DNA SIZE OF DNA PURITY OF DNA

3. 1.ABSORPTION of light by dna DNA absorbs light in UV light of electromagnetic spectrum. UV ranges from 200 to 400 nm The absorption maxima is 260 nm (absorption maxima – It is the point at which the biomolecule absorbs the maximum radiation). This absorption can be monitored by using a spectrophotometer and calculated by Beer Lamberts Law. This method can be used to determine the concentration of DNA. More the DNA present ,higher the absorption . The less ordered the bases the more ultraviolet light is absorbed. Free bases absorb 1.60 units at 260 nm. Single stranded DNA absorbs 1.37 units at 260 nm Double stranded DNA absorb 1.00 units at 260 nm

4. 2.BUOYANT DENSITY OF DNA. Also called the density of DNA. it is determined by isopycnic centrifugation. In this method a salt is used cesium chloride ( Cscl ). The DNA is mixed in this solution and it is spun at high speed, so the DNA at this solution at a particular band according to its density. The buoyant density of DNA is 1.73 g/cm^3 =6 M Cscl Buoyant density of DNA is used to estimate the G +C content upon centrifugation , will form a density gradient with most dense solution at the bottom More dense DNA will migrate downward and less dense DNA upwards forming bands G C base pairs is more dense than A T base pairs because of the presence of 3 hydrogen bond therefore DNA with more G C base will migrate towards the lower portion and DNA with A T base pairs at the top.

5. 3.DENATURATION OF DNA DNA is considered to be denatured when the double stranded DNA molecule is converted into two single stranded molecules ,which occurs when the hydrogen bonds between the strands are broken FACTORS AFFECTING DENATURATION 1) TEMPERATURE As thermal energy increase , the frequency of hydrogen bonds breaking between the molecule increases and the DNA will separate into single strands. The temperature at which half of the DNA molecule denature is called Melting Temperature (Tm) Tm is greater for DNA s with higher G C content while DNA with high A T base content has less Tm. 2) pH ACIDS ; In neutral pH ,DNA molecules are stable. At lower pH result in the breakage of phosphodiester bonds between nucleotides and breakage of N- glycosidic linkage between the sugars and purines ( A ,G) ALKALI ; At pH 9 or higher DNA is suspectable to alkaline denaturation and all the hydrogen bonds are disrupted .

6. DENATURATION OCCUR IN TWO WAYS ; PHYSICAL AND CHEMICAL DENATURATION Physical denaturation is done by heating . Usually, high temperature above 90 degree Celsius result in the denaturation of DNA. By providing enough kinetic energy can breakdown hydrogen bonds between the base pairs which results in the separation of double stranded into single strands . The degree of denaturation can be determined by spectrophotometer by monitoring the absorbance of light at 260 nm. The UV absorbance increases DNA is denatured , this phenomenon is called as hyperchromic shift. Double stranded DNA absorb less UV light but increase when the strands separate ( here the base –base interaction is reduced)

7. Chemical denaturation Denaturation in the presence of chemical agents, such as urea and formamide ,accelerates denaturation by stabilizing purines and pyrimidines. Thus ,they decrease the Tm . 95% of formamide completely denatures DNA at room temperature . Various concentration of sodium hydroxide and dimethyl sulfoxide (DMSO) also decrease the Tm .

8. 4.renaturation Renaturation of DNA is the process of annealing the two strands of two complimentary strands od DNA. Eventually ,it occurs on cooling. Renaturation happens through the reformation of hydrogen bonds between complementary base pairs , which in turn the DNA strands together to form the double stranded DNA. Renaturation rates dependent on the concentration of DNA molecules ,the more molecules of complimentary DNA molecules present faster they can find each other . On renaturation UV absorbance decreases.

9. 5.Solubility of dna DNA is soluble in water because of the presence of highly charged phosphate backbone. RNA is more soluble in aqueous solutions than DNA because ribose has a 2’OH group where deoxyribose contains 2’H . 6.SIZE OF DNA Electrophoresis DNA is negatively charged because of the presence of negatively charged phosphate. when DNA is placed in an electric field it will migrate towards the positively charged electrode . If DNA is electrophoresed through a gel , smaller piece will migrate faster than larger piece, Agarose gel is used to separate larger DNA molecules and PAGE is used to separate small piece of DNA . The size of DNA also can be determined by electron microscopy.

10. 7.Purity of dna To evaluate the purity of DNA ,measure the absorbance from 260 nm to 360 nm . The purity calculation is the ratio of absorbance at 260 nm divided by the reading at 280 nm. A good quality of DNA have this ratio ~1.7 -2.0 If the ratio is lower indicate the presence of proteins ,phenol or other contaminants that absorb strongly at or near 280 nm.

11. THANKU KESSIYA T PETER