In sitedirected mutagenesis restriction enzymes cut out a short sequence of nucleotides that is then replaced by a synthetic mutated DNA sequence

Plasmid contains gene with mutated sequence

Mismatched bases

Target sequence

Mismatched bases ft The oligonucleotide and the target DNA sequence pair.

The oligonucleotide is a primer for DNA synthesis,.

.which produces a molecule with a single mismatched pair.

^ The DNA is transferred to bacterial cells, and the mismatched bases are repaired by bacterial enzymes.

^ About half of the plasmids will have the original sequence and the other half will have the altered sequence.

Plasmid with original sequence

Plasmid with desired sequence

Target sequence

An oligonucleotide is created that differs from the target sequence in a single nucleotide.

Plasmid with original sequence ft The oligonucleotide and the target DNA sequence pair.

The oligonucleotide is a primer for DNA synthesis,.

.which produces a molecule with a single mismatched pair.

^ The DNA is transferred to bacterial cells, and the mismatched bases are repaired by bacterial enzymes.

^ About half of the plasmids will have the original sequence and the other half will have the altered sequence.

Plasmid with desired sequence yn The bacteria are then screened for the presence of the altered gene.

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