Chemistry

Genetic engineering methods

Genetic engineering methods


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The lysis of the bacterial cells

The isolation of the DNA begins with the lysis of the bacterial cells, which can be obtained from a culture with the highest possible cell density by centrifugation and then resuspended in an appropriate cell disruption buffer. While the small circular plasmids are released relatively quickly from bacterial cells, the chromosomal DNA breaks easily when shear forces occur in the insulation.

Gentle cell disruption is achieved through the use of enzymes, while mechanical processes such as ultrasound treatment, breaking cells in the French press or the glass bead mill are generally not recommended for DNA isolation, as large DNA molecules are fragmented in these processes.

A. The enzymatic digestion

For the isolation of genomic DNA, bacterial cells are preferably disrupted with a combination of enzymes such as lysozyme and proteinase K. Lysozyme is an enzyme found in chicken egg white or tear fluid that binds the glycosidic bonds betweenN-Acetylmuramic acid (NAM) andN-Acetylglucosamine (NAG) hydrolyzed in the murein sacculus of the bacterial cell wall. Proteinase K is a serine protease that specifically cleaves peptide bonds in which one of the two amino acids involved has an aromatic, aliphatic or hydrophobic character. This enzyme works optimally at 55-60 ° C and in the presence of 0.5% SDS, so that the cytoplasmic membrane of the bacterial cells ruptures during incubation in proteinase K buffer.

B. The digestion by detergents

Detergents are primarily used to obtain the plasmid DNA. There are various methods for cell disruption with detergents, which are used depending on the type and use of the plasmid DNA to be isolated:

  • The minilysate method or alkaline lysis is the most common method for isolating plasmid DNA, which can also be used for large and lowcopy-Plasmid is very suitable. Here the cells are treated with a combination of sodium dodecyl sulfate (SDS1)) and NaOH digested.
  • During cooking lysis, the bacterial cell walls are destroyed by lysozyme and the lysed bacteria are boiled for a short time. The bacterial cell residues (debris) are removed from the lysate by centrifugation and the plasmid DNA is then precipitated.
  • The non-ionic detergent Triton X-100 can also be used to disrupt bacterial cells in the presence of high concentrations of lithium chloride. Following a lysozyme treatment, this so-called lithium method leads to the dissolution of the bacterial cytoplasmic membrane. After the proteins have been completely denatured by phenol / isoamyl alcohol, the plasmid DNA is released. However, this method is not suitable for isolating larger plasmids.



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