Features of cloning vectors

This article offers a review of what a good cloning vector is, and what features a good vector should have.

Cloning vectors are DNA molecules into which foreign DNA can be inserted. Typically, scientists adapt naturally occurring structures that can replicate independent of chromosomal DNA. For example, plasmids are circular, double-stranded DNA molecules (see image below) that can be found in several numbers within a single bacterial cell.

This kind of plasmid can be modified to include genes that are useful for cloning and manipulating other genes. A modified plasmid that is used for cloning is known as a vector. A successfully modified vector becomes a recombinant, or recombinant plasmid.

The image below represents a cloning vector.

Why does it look so different from the plasmid? A vector is also double-stranded, but is usually represented in a simplified manner to make it easy to understand. In the above image, the double-stranded 'backbone' of the vector is depicted as a thin black circle, and important features and genes are depicted by the colored segments.

Let's take a closer look at some of the features that all cloning vectors require.

The ori, or origin of replication, is the sequence at which replication of DNA begins. This sequence is also linked to the copy number of the vector, and so controls how many times your gene of interest will be produced in the host cell.

Selectable markers are genes that help identify bacteria that have successfully transformed, or taken up the recombinant plasmid. Antibiotic resistance genes are commonly used markers.

All cloning vectors need sites that will allow the insertion of foreign DNA into it. Most commonly, this involves the use of restriction enzyme recognition sites. Ideally, the enzyme should have $1$‍, or a maximum of $2$‍ recognition sites, preferably within the selectable marker sequences (as seen in the image below).

Cutting the vector within the selectable marker will interrupt that gene, and allow for identification of recombinants. For example, using a cloning site within an ampicillin resistance gene will result in recombinants that are no longer resistant to that antibiotic.

Alternately, we can also put in new markers using cloning sites, and thus screen for recombinants by looking for the new gene.

Let's do a quick quiz on what a good cloning vector should and shouldn't have.