Molecular cloning

Molecular cloning refers to the assembly of DNA molecules into a vector and the subsequent transformation of an organism (often bacteria or yeast). Molecular cloning methods are central to biology and medicine. The term molecular cloning comprises many different techinques. A typical molecular cloning flow looks as follows:

  1. Isolation of the gene of interest

  2. Cloning the gene into a vector

  3. Transforming the host organism with the vector construct

  4. Antibiotic selection of transformed cells

  5. Isolation of clones with the same genetic background

Confirmation of plasmid assembly can be determined by performing DNA sequencing. It is important to confirm that the inserts have been successfully ligated into a plasmid vector with the correct conformation and reading frame. Frame shift can cause nonsense or misense mutations that lead to the expression of nonfunctional proteins.