Applied Biology - Class 12
- DNA cloning and recombinant DNA
- A brief history of restriction enzymes
- Restriction enzyme mechanism
- Parts of a cloning vector
- Features of cloning vectors
- Multiple cloning sites & restriction enzymes
- Insertional inactivation (two antibiotic selectable markers)
- Insertional inactivation using Lac Z gene (Blue white screening)
- Competent cells, transformation, and other methods of DNA delivery
- Cloning sites and insertional inactivation
What does the ideal cloning vector look like? Ori, selectable markers, and more.
This article offers a review of what a good cloning vector is, and what features a good vector should have.
|plasmid||circular DNA found in bacteria; can replicate independent of chromosomal DNA|
|vector||modified plasmid that can be used to clone and express foreign DNA|
|ori||origin of replication; site from which DNA replication starts|
|selectable marker||gene in vector that enables selection of recombinants|
|cloning site||restriction enzyme recognition site (often within selectable marker); site at which foreign DNA is ligated|
What's a cloning vector?
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.
Origin of replication
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
, or a maximum of 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.
Copy number of plasmids
Plasmids with very low copy numbers (
- ) are usually not ideal for cloning purposes.
Which of the following parts of the vector might contribute to the low copy number?
Not all cloning sites are within selectable markers. Imagine that you have a vector that will continue to express a gene after succesful transformation. In other words, inserting foreign DNA into this vector will not affect this selectable marker.
Which of the following options would make good selectable markers for this vector?
A cloning vector has
genes. The number of EcoRI recognition sites within each gene are listed in the table below.
|gene 1||gene 2||gene 3|
Which gene would you choose to ligate foreign DNA into?
Want to join the conversation?
- can we say that recombination precedes transformation?(3 votes)
- Yes, the foreign DNA is inserted into the vector (recombination), after which the vector is incorporated into the host cell for replication (transformation).(4 votes)