lecture 4- site directed mutagenesis in biologics

What is site directed mutagenesis

a targeted and specific change in a gene

Type of mutations used

Point mutations (silent, nonsense, missense)

Sickle Cell Anemia point mutations

A single base change in the beta-globin gene alters hemoglobin structure.

Kunkel's Method

o Uses bacteriophage (virus that infects bacteria) to carry the gene of interest. A mutated DNA fragment is inserted into the virus. The virus infects bacteria, where enzymes help incorporate the mutation into the DNA

Cassette Mutagenesis

o A small piece of DNA (cassette) containing the mutation is created. The gene is placed inside a plasmid (circular DNA in bacteria). A restriction enzyme (DNA scissors) cuts the gene at a specific site. The mutated cassette is inserted, replacing the original sequence.

PCR-Based Mutagenesis

Modification of primer causes mutation at specific site in PCR

In Vivo Mutagenesis

The process of introducing mutations inside living cells using mutator strains (CRISPR/Cas9)

CRISPR/Cas9

a revolutionary gene editing technique derived from the immune system of simple prokaryotes

Common Procedure

- Identify the gene of interest.

- Design a primer with the desired mutation.

- Amplify using PCR and introduce it into bacterial cells.

Select positive clones and confirm the mutation through sequencing

tenecteplase (TNK-tPA)

- Modified tissue plasminogen activator (tPA) with improved clot-busting properties.

Insulin modifications; Insulin Glargine

A21 Asparagine → Glycine

insulin Lispro modification

B28 Proline ↔ B29 Lysine.

Insulin Aspart. modification

B28 Proline → Aspartic Acid

Liver Fatty Acid Binding Protein (L-FABP) mutation

Threonine (T) → Alanine (A).

1. Primer Design Considerations

- Mutation should be centrally located in the primer.

- GC content should be at least 40%.

- Be at least 24-45nucleotides long

- Melting temperature (Tm) should be ≥ 78°C.

- 3' end should end with C or G

1. Basic Mechanism

- A primer with the desired mutation binds to the target DNA.

- DNA polymerase extends the primer, creating a mutated DNA strand.

- The mutated DNA is introduced into host cells for replication and expression