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Powerpoint Presentation: Gel Electrophoresis
Powerpoint Presentation: DNA Size
Powerpoint Presentation: Molecular Genetics

 

Molecular Genetics Index

The Chemical Nature if the Gene
The Nucleic Acids
The Gene
Ribonucleic Acid (RNA)
The Characteristics of the Genetic Code
The Genetic Code
Gene Expression
Translation
The lac Operon
Gene Mutations
Genetic Engineering

Topic Chapters Index

 

MOLECULAR GENETICS

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Electrophoresis

The migration of electrically charged particles in colloidal solution towards the oppositely charged electrode.

Similar to electrolysis:

Electrolysis

For large charged particles the rate of migration towards the electrode will depend upon the size of the charge carried in relation to the mass of the particle and the magnitude of the current.

 

Paper electrophoresis of amino acids

Paper electrophoresis of amino acids

 

At pH 7 basic amino acids will be positively charged and so they will behave as cations. Acidic amino acids will be negatively charged and so they will behave as anions. Neutral amino acids will be both positively and negatively charged (zwitterions) and so they will stay in the centre. These amino acids are said to be at their isoelectric point. Each type of amino acid has its isoelectric point at a particular pH, it depends upon the number of ionisable groups in the molecule. At any pH above the isoelectric point the molecule will have a net negative charge and move towards the anode. Similarly at pHs below the isoelectric point the molecule will have a net positive charge and move towards the cathode.

The relative charge on each species of amino acid will determine the speed and direction of travel. Thus they can be separated.

 

Electrophoresis of proteins

As proteins are made of amino acids and amino acids migrate in an electric current, then proteins too will migrate (slowly). The speed and direction of migration will depend upon the numbers of the characteristic amino acids in the protein's structure. A protein made up of a lot of basic amino acids will not behave in the same way as a protein made up of acidic ones, they will have different isoelectric points.

Even small differences are detectable e.g. the difference between normal and sickle cell haemoglobin where there is a difference of only one amino acid.

 

Gel electrophoresis of proteins and DNA

The process of electrophoresis of large molecules such as proteins and nucleic acids is carried out on gels. Here the correct buffering conditions can be carefully controlled and the effects of vibrations and convection currents are removed. Furthermore, the pH of the gel can be varied along its length to provide a pH gradient as well as an electrical gradient. This permits high resolution separation of very small samples.

Proteins a separated on polyarylamide gels; The spaces between the matrix of these gels can be chosen to suite the proteins being studied. The proteins are treated with a detergent, SDS, so that they separate into their subunits and uncoil into long polypeptide chains.

DNA is analysed on agarose gels which have bigger spaces to allow larger molecules of DNA to separate. DNA does not need to be treated with a detergent as it is not folded up.

Each nucleotide on the DNA molecule carries a negative charge. Therefore, DNA molecules only move from the negative cathode to the positive anode.

As the negative charge increases with size, big DNA molecules move more quickly. However, bigger molecules move more slowly through the gel. The result is a steady and fine separation of DNA molecules by size. Molecules which differ by only one nucleotide in their length can be separated.

The analysis of the gel after the proteins or DNA have been separated is carried out by staining, spectroscopy or autoradiography in the case of labelled molecules. This reveals bands in the gel where the proteins or DNA fragments are located. Reference proteins are run in the gel at the same time so that the molecules in the mixture can be identified.

Gel electrophoresis

 

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