The Denaturation Melting of DNA Is Used to Analyze Its Structure

The double-stranded structure of DNA can be separated into two component strands (melted) in solution by increasing the temperature or decreasing the salt concentration. Not only do the two stacks of bases pull apart but the bases themselves unstack while still connected in the polymer by the phosphodiester backbone. Concomitant with this denaturation of the DNA molecule is an increase in the optical absorbance of the purine and pyrimidine bases—a phenomenon referred to as hyperchromicity of denaturation. Because of the

Minor groove

Major groove <

Minor groove

Major groove <

34 A

20 A

Figure 35-2. A diagrammatic representation of the Watson and Crick model of the double-helical structure of the B form of DNA. The horizontal arrow indicates the width of the double helix (20 A), and the vertical arrow indicates the distance spanned by one complete turn of the double helix (34 A). One turn of B-DNA includes ten base pairs (bp), so the rise is 3.4 A per bp. The central axis of the double helix is indicated by the vertical rod. The short arrows designate the polarity of the antiparallel strands. The major and minor grooves are depicted. (A, adenine; C, cytosine; G, guanine; T, thymine; P, phosphate; S, sugar [deoxyribose].)

34 A

20 A

Figure 35-2. A diagrammatic representation of the Watson and Crick model of the double-helical structure of the B form of DNA. The horizontal arrow indicates the width of the double helix (20 A), and the vertical arrow indicates the distance spanned by one complete turn of the double helix (34 A). One turn of B-DNA includes ten base pairs (bp), so the rise is 3.4 A per bp. The central axis of the double helix is indicated by the vertical rod. The short arrows designate the polarity of the antiparallel strands. The major and minor grooves are depicted. (A, adenine; C, cytosine; G, guanine; T, thymine; P, phosphate; S, sugar [deoxyribose].)

stacking of the bases and the hydrogen bonding between the stacks, the double-stranded DNA molecule exhibits properties of a rigid rod and in solution is a viscous material that loses its viscosity upon denaturation.

The strands of a given molecule of DNA separate over a temperature range. The midpoint is called the melting temperature, or Tm. The Tm is influenced by the base composition of the DNA and by the salt concentration of the solution. DNA rich in G-C pairs, which have three hydrogen bonds, melts at a higher temperature than that rich in A-T pairs, which have two hydrogen bonds. A tenfold increase of monovalent cation concentration increases the Tm by 16.6 °C. Formamide, which is commonly used in recombinant DNA experiments, destabilizes hydrogen bonding between bases, thereby lowering the Tm. This allows the strands of DNA

NN N

Thymidine

Thymidine

Adenosine

Cytosine \

Cytosine \

H Guanosine

Figure 35-3. Base pairing between deoxyadenosine and thymidine involves the formation of two hydrogen bonds. Three such bonds form between deoxycytidine and deoxyguanosine. The broken lines represent hydrogen bonds.

H Guanosine

Figure 35-3. Base pairing between deoxyadenosine and thymidine involves the formation of two hydrogen bonds. Three such bonds form between deoxycytidine and deoxyguanosine. The broken lines represent hydrogen bonds.

or DNA-RNA hybrids to be separated at much lower temperatures and minimizes the phosphodiester bond breakage that occurs at high temperatures.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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