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DNA structure o Deoxyribonucleic acid (DNA) : Double helix, Polymeric molecule , unit of heredity & organized into genes , contains genetic information.o Made up of nucleotides.

DNA structure
• Deoxyribonucleic acid (DNA) : Double helix, Polymeric molecule , unit of heredity & organized into genes , contains genetic information.
• Made up of nucleotides.
• 3 parts of nucleotide:

1. 5 carbon sugar; deoxyribose


2. phosphate group


3. nitrogen base (A, T, G,C)
   Base pairing:
   • Each side of the DNA strand forms a "complementary" hydrogen bond to the other side.
   • A must always opposite to a T.
   • C must always opposite to a G.
   • There is hydrogen bonding between the bases.
   • 2H-bonds between A and T and 3H bonds between G and C.
   Chargaff's rule:
   • The concentration of thymine was always equal to the concentration of adenine
   (A = T).
   • The concentration of cytosine was equal to the concentration of guanine (G=C).
   • This strongly suggest that thymine and adenine as well as cytosine and guanine were present in DNA with fixed interrelationship.
   • Also the total concentration of purines (A +G) always equal to the total concentration of pyrimidine (T +C).
   • All DNA possess purine and pyrimidine in equal proportions (1:1 ratio).
   • However, the (T+A)/ (G+C) ratio was found to vary widely in DNAs of different species.
   • lf (A +T) > (G + C) then DNA is referred as (AT- type).
   • lf (G + C) > (A +T) then DNA is referred as (GC- type).
   Example:
   • If A = 20% in particular double stranded DNA molecule , what are the percentage % of T ,G, C?
   SOLUTION:
   If A is 20% then according to Chargaff's rule T will be also 20% because they must equal A=T So A + T are 20% + 20% = 40% .
   Assuming 100% ,subtract the A & T amounts from 100.
   ( 100% – 40% )= 60%,
   60% is the amount of C & G together , so 60% / 2 = 30% So, C is present 30% and G is present 30%.
   Denaturation/ Melting:
   • Loss of helical structure of DNA called denaturation or melting.
   • Change from double strand DNA to single strands.
   • In living cell, DNA denature or separates during replication by helicases .
   • In laboratory, DNA strands can be separated by:

1. change pH – alkaline.


2. Heat – increasing the temperature, increases the denaturation.
   How to measure the denaturation/ melting?
   • Denaturation measured by spectrophotometer.
   • The purine and pyrimidine bases in DNA strongly absorb ultraviolet light.
   • Double-stranded DNA absorbs UV light less strongly than denatured DNA due to the stacking interactions between the bases.
   • The phenomenon of UV absorbance increases as DNA is denatured is known as the hyperchromism.
   UV Absorbance Melting Curve
   • Nucleic acids display an ultraviolet absorption peak at 260 nm and their absorbance at 260 nm (A260) increases when denaturation or melting occurs.
   • The data curve acquired when measuring UV absorbance of nucleic acid ( A260) with respect to the increase in temperature is called a UV absorbance melting curve.
   Melting temperature (Tm ) of DNA
   • Tm of DNA: Temperature at which half of the DNA molecules are denatured.

• i.e. the ratio of the double strands to the single strands becomes equal.
  UV Absorbance Melting Curve
  • To determine Tm from the melting curve, as shown in the figure, the baseline is determined with respect to the pre transition region and post transition region.

UV Absorbance Melting Curve
• Next, the median of the 2 baselines is drawn, and the temperature at which this line intersects with, the melting temperature(Tm) is obtained.

Factors affecting Tm of DNA:

1. Nucleotide content of DNA molecule.


2. Length of DNA molecule.


3. Ionic strength of the DNA solution.


4. Nucleotide content of DNA molecule
   • In DNA, A pairs with T with 2 hydrogen bonds, G pairs with C with 3 hydrogen bonds.
   • G C base stacking interactions are more stable compared to A T base.
   • Tm of DNA is greatly influenced by GC content of nucleotide.
   • Example:
   1.• GC Rich
   • Need higher energy to disrupt the double helix.


5. • AT Rich
   • Need lower energy to disrupt the double helix.

• Strand which contain more Guanine and cytosine will be more stable and will need higher Tm to be denatured to single strands.
• Higher the GC content = higher the Tm of DNA.

2. Length of DNA molecule
   • More the length, greater the stabilizing forces between two DNA strands.
   • Longer the length of DNA molecule = higher the Tm of DNA.


3. Ionic strength of the DNA
   • Each phosphate group in the DNA double strand carry negative charges, these charges make the DNA molecule unstable.
   • Inside the eukaryotic cell, Histones play a role in DNA stability, it has a positive charge which neutralize the negative charge on DNA molecule.
   • In the laboratory, positive ions (e.g. Na+) stabilize the DNA molecule in a solution by neutralizing the negative charge on DNA molecule.
   • The Ionic strength = the total ion concentration in the DNA solution.
   • Higher the ionic strength of solution tends to higher Tm of DNA.

Renaturation:
• Also known as annealing .
• Separated complementary strands of DNA can spontaneously re-associate to form double helix.
• Temperature of DNA lowered below its melting temperature, rewinding of DNA takes place.
• So, DNA can melt and reanneal itself reversibly.
• Absorbance decreases and viscosity of the solution increases, this phenomenon is called hypochromism.
Experiment: To Determine DNA Tm
Principle:
• When a dilute aqueous DNA solution is heated slowly, the two strands of the double helix gradually separate, leading to the formation of a single stranded DNA (denaturation) .
• This results in an increase in absorbance at 260 nm.
• Temperature for midpoint of denaturation gives Tm by increasing the temperature slowly and measuring absorbance at 260 nm as melting profile can be generated.
• The DNA of each species has a specific denaturation curve which is dependent on the %GC content, length, and ionic strength.

Procedures
1.In test tube, prepare 1ml of the sample by diluting your extracted DNA (your stock) to 10μg / ml buffer.
2.In another test tube, pipette 2 ml of distilled water (blank).
3.Cover the test tubes of the sample and the blank with aluminum foil.
4.Place the tubes into a water bath at 25°C and allow temperature to equilibrate (5min).
5.Immediately, transfer the blank and sample into quartz cuvette then read the absorbance at 260nm.
6.Raise the temperature of the water bath to 50°C, 60°C, 70°C, and boiling, then repeat step 4 and 5.
7.Draw a graph between Temp. on X axis and absorbance at 260nm on Y axis.
Results
• As the temperature increase, as the A260 increase.
• The temperature for midpoint of denaturation gives Tm.