Chemistry

Genetic engineering methods

Genetic engineering methods


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Gel electrophoresis: introduction

Gel electrophoresis is a biochemical separation process in which the migration of charged molecules in an electric field is used to separate them.

Nucleic acids are polyanions, i.e. they are negatively charged due to the sugar-phosphate backbone. In the electric field, polyanions migrate to the anode, and the faster the smaller they are. In gel electrophoresis, agarose is usually used as the matrix, and in the case of very small nucleic acids, polyacrylamide is also used. The pore size of the matrix determines how quickly a nucleic acid of a certain size can migrate to the anode. With longer fragments, there is greater friction than with shorter fragments, so they are retained proportionally more strongly and therefore migrate more slowly. In this way, not only is a separation of the molecules achieved in gel electrophoresis, but the length of a DNA or RNA fragment compared to a standard with a known fragment size can also be calculated on the basis of the relative migration distance.

In contrast to proteins, the relationship between charge and size (in base pairs) is constant in nucleic acids. For linear double-stranded DNA fragments, there is a linear dependence between the decadic logarithm of the fragment length (in base pairs) and the relative migration distance (in cm, based on the entire migration route).

The separation must take place in a low-salt environment, as this is the only way to ensure that the electrical field strength acts homogeneously on the nucleic acids. The migration behavior of non-linear DNA cannot be compared to linear standard DNA, since circular DNA behaves completely differently in the agarose gel.



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