17: Molecular Cloning
Molecular cloning refers to the isolation and amplification of a specific DNA sequence in vivo . It uses a host organism (usually bacteria) to produce a large number of identical copies of a recombinant DNA sequence that can be used for other molecular biology applications.
Also known as
Cloning
Samples needed
Concentrated solutions of a cloning vector (plasmid) and DNA containing the sequence to be cloned. The vector needs to contain an origin of replication specific to the organism into which the vector will be transformed, along with a gene encoding a selectable marker (often an antibiotic resistance gene).
Method
There are many methods for cloning a piece of DNA, but we will focus here on a classical molecular cloning process using restriction enzymes. The principles apply to most other cloning methods. You can see a diagram depicting the steps to be described in Figure 7.1 in J.C. Lessard's chapter "Molecular Cloning." [1] First, the DNA to be cloned (called the insert) is obtained and isolated through PCR, DNA synthesis, or an alternative method. The vector and insert are then digested with restriction enzymes, producing ends that are complementary to each other. The digested DNA is purified. The digested vector and insert are incubated with DNA ligase and transformed into a host (usually bacteria). Host cells that take up the plasmid are selected for by plating on agar plates containing a chemical/compound that is toxic to cells lacking the plasmid. Cell colonies that grow on the plates can be expanded and the plasmid DNA can be purified from them. Restriction enzyme digestion (see interpretation), PCR, or DNA sequencing is often used to determine which plasmids contain the desired insert DNA.
Controls
Typically, a ligation reaction containing only the vector + DNA ligase is included, to make sure that bacterial colonies recovered after transformation don’t contain a religated vector without an insert.
Interpretation
In this cloning experiment, alkaline protease genes from two haloalkaliphilic bacteria strains isolated in India (OK-5 and OM-6) were PCR amplified using genomic DNA from the bacteria as a template. The PCR products and the pET21a+ vector were both digested with Bam HI and Sal I restriction enzymes and the products were purified. Following ligation, transformation into E. coli, and plating on ampicillin-containing solid media, colonies were isolated, grown in ampicillin-containing liquid media, and plasmids were purified from each culture.
Purified plasmids were then digested with Bam HI and Sal I restriction enzymes and the products were separated by gel electrophoresis. The results from lanes 2 and 3 show that the alkaline protease gene from the OK-5 strain was successfully cloned into pET21a+, while the results from lanes 4 and 5 show that the alkaline protease gene from the OM-6 strain was successfully cloned into the plasmid.
Image Descriptions
Figure 1 image description:
A map of the pET21a+ cloning vector. Unique cloning sites are indicated by lines labeled with the corresponding restriction enzyme recognition sequences. ↵
Figure 2 image description:
An agarose gel, stained with the intercalating agent ethidium bromide, showing the restriction enzyme digestion products of recombinant vectors created in a cloning experiment. Plasmids containing an insert produce two bands on the gel. ↵
Thumbnail
"BamHI.png" ↗ by Simon Caulton is licensed under CC BY-SA 4.0 ↗.
Image description: Restriction of DNA by BamHI enzyme. The enzyme cuts the DNA at the sequence GGATCC and leaves 'sticky ends' that can attach to complementary sticky ends.