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.
