DNA melting and Buoyant density

Denaturing and Annealing of DNA:

The DNA double strand can be denatured if heated 95 degree Celsius or treated with chemicals. AT regions denature first and then GC regions because of the three hydrogen bonds in between GC. DNA denaturing is a reversible process, as DNA strands can be re annealed if cooled. This process can be monitored using the hyperchromicity(melting profile).

Hyperchromicity:

It is used to monitor DNA denaturation and annealing. It is used on the fact that single stranded DNA gives higher absorption reading than double stranded at wavelength 260nm.

Denaturation: 

The strands of the DNA double helix are held together by hydrogen bonding interactions between the complementary base pairs. Heating DNA in solution easily breaks these hydrogen bonds, allowing the two strands to separate this process is known as denaturation or melting of DNA.

The two strands may re associate when the solution cools, reforming the starting DNA duplex this process is known as renaturation or hybridization.

These process form the basis of many important techniques for manipulating DNA. for example a short piece of DNA called an oligonucleotide can be used to test whether a very long DNA sequence has the complementary sequence of the oligonucleotide embedded within it. 

Using hybridization a single stranded DNA molecule can capture complementary sequences from any source. Single strands from RNA can also re associate. DNA and RNA single strands can form hybrid molecules that are even more stable than double stranded DNA. These molecules form the basis of a technique that is used to purify and characterize messenger RNA molecules corresponding to single genes.

Ultraviolet Absorption:

DNA melting and re association can be monitored by measuring the absorption of Ultraviolet(UV) light at a wavelength of 260 nanometer(billionths of a metre). When DNA is in a double stranded conformation, absorption is fairly weak, but when DNA is single stranded, the un stacking of the bases leads to an enhancement of absorption called hyper chromocity(melting profile). Therefore, the extent to which DNA is single stranded or double stranded can be determined by monitoring UV absorption.

The melting temperature of DNA depend on

1. Content of G C base pairs
2. Size of DNA
3. Ph
4. Ionic strength.

Buoyant Density of DNA

It is the density of the solution at which the DNA feels no net force during centrifugation is called buoyant density.

This is the density in the density gradient where that particular DNA molecule will form bands as it stops going up or down. Under constant conditions(usually 25 degree Celsius in ceasium chloride at neutral Ph) the buoyant density of DNA is related to the GC content.

     %G+C content=buoyant density-1.660 X 100/0.098

The fraction of Phaseolus aerus DNA has a buoyant density of 1.695 g/cubic centimeter and that of E.coli DNA has a buoyant density of 1.710 g/ cubic centimeter in cesium chloride. Most nuclear DNAs from higher plants have buoyant density within the range 1.69-1.71 g/cubic centimeter. However the presence of 5-methyl cytosine serves to reduce the density slightly, thereby giving rise to an underestimate of the GC content. In general, 1% methylation decreases the buoyant density by 1 mg/cubic centimeter. Certain sequences of bases may also distort the relationship between the base composition and buoyant density. Furthermore, ssDNA is denser than dsDNA of similar base composition by approximately 0.015 g/ cubic centimeter and under alkaline conditions the density is increased by 0.06 g/cubic centimeter.

Factor affecting Buoyant Density

• Buoyant density of DNA depends on the fallowing factors:
• Nature of the cesium chloride,
• Presence of heavy metals or DNA binding dyes,
• The Ph, and 
• The temperature