Molecular Cloning Methods (CMB part 2)

To clone a gene, it must be inserted into a vector capable of carrying the gene into a host cell and ensuring its replication. This insertion is typically achieved by cutting both the vector and the target DNA with the same restriction endonucleases to create matching “sticky ends.” Cloning vectors in bacteria are primarily categorized as plasmids or phages. Plasmid cloning vectors include pBR322 and the pUC plasmids. The pUC plasmids and pBS phagemids facilitate convenient screening, as they possess an ampicillin resistance gene and a multiple cloning site that disrupts a partial β-galactosidase gene. The resulting clones are resistant to ampicillin and lack active β-galactosidase, which is easily identifiable through a color test. Two prominent types of phage vectors are widely used in cloning. The first is λ (lambda), which has had nonessential genes removed to accommodate inserts, allowing for the insertion of up to 20 kb. Cosmids, combining features of phage and plasmid vectors, can accept inserts up to 50 kb, making them ideal for constructing genomic libraries. The second major type is M13 phages, which offer a multiple cloning region and the ability to produce single-stranded recombinant DNA. This single-stranded DNA is particularly useful for sequencing and site-directed mutagenesis. Phagemids, plasmids with an origin of replication for single-stranded DNA phages, can also generate single-stranded copies of themselves. Expression vectors are specifically designed to maximize the production of a protein encoded by a cloned gene. Bacterial expression vectors optimize expression by incorporating strong bacterial promoters and ribosome-binding sites, which are typically absent in cloned eukaryotic genes. Most cloning vectors are inducible to control protein production efficiently.

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