6.5 Mutagenesis

Mutagenesis is the process of introducing changes into the DNA sequence. It is used in studies of gene, and the resulting protein, function. Mutations may be:

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Random mutagenesis can make use of DNA shuffling or error prone polymerases. DNA shuffling: the DNA is digested randomly using DNaseI, denatured and used in PCR with no primers.

Figure showing how a DNA sequence can be mutated using PCR with no sequence information needed
Figure 20: DNA Shuffling: Random mutagenesis by PCR. The DNA is digested at random using DNaseI. The resulting fragments are denatured and used in PCR. At the annealing stage random complementary fragments bind.

Error-prone Polymerase: This method relies on the fact that Taq polymerase has no proof-reading abilities. Other changes are the use of an error-prone buffer in the reaction which has increased magnesium ion concentration and the added manganese ions. Altering the Mn2+ concentration will change the number of mutations per kilobase. The concentration of dTTP is increased and dATP decreased.

Figure showing the requirements for error prone polymerase mutagenesis to work.
Figure 21: Error prone polymerase mutagenesis. PCR is carried out using buffers which differ as shown.
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