The process of molecular cloning is a fundamental technique in biology that allows scientists to create identical copies of a specific DNA sequence. This replication enables the study of genes and the production of useful proteins, such as insulin or vaccines, in large quantities. At its core, cloning involves combining two primary components: the gene of interest, known as the insert, and a small, circular piece of DNA called a plasmid, which acts as a vehicle or vector. This combined structure, known as recombinant DNA, can be replicated independently inside a host cell, typically a bacterium. The following steps detail the sequential process required to successfully merge these elements and amplify the desired gene.
Preparing the Gene and Plasmid DNA
The first procedural step in cloning involves precisely preparing both the gene insert and the plasmid vector so they can be joined together. This preparation relies on a class of enzymes called restriction endonucleases, which act as molecular scissors by recognizing and cutting DNA at specific, short nucleotide sequences. The goal is to cut the circular plasmid to make it linear and to excise the gene insert from its original source, ensuring the resulting ends are compatible for joining.
Restriction enzymes often create staggered cuts, leaving short, single-stranded overhangs known as “sticky ends,” which are highly desirable for cloning because they are complementary and can easily anneal to each other. Alternatively, some enzymes cut straight across the DNA double helix, producing “blunt ends” that lack overhangs and are universally compatible with any other blunt end. Using the same or compatible restriction enzymes on both the plasmid and the insert ensures that the resulting ends match, promoting the correct orientation of the insert in the vector.
After the DNA is cut, the fragments are separated and purified using gel electrophoresis, a technique that uses an electric current to sort DNA pieces by size through a gel matrix. The desired DNA bands are then physically excised from the gel and cleaned up. This process removes impurities that could interfere with the subsequent joining reaction.
Joining the DNA Fragments
Once the gene insert and the linearized plasmid have been isolated and purified, the next step is the chemical reaction of assembly, known as ligation. This process requires the enzyme DNA ligase, which functions as a molecular glue to covalently link the insert into the plasmid backbone. The ligase creates a phosphodiester bond between the 3′-hydroxyl group of one DNA fragment and the 5′-phosphate group of the other, effectively sealing the nicks in the DNA backbone and making the plasmid circular again.
The ligation reaction is set up in a small volume by mixing the purified gene insert, the linearized plasmid vector, DNA ligase, and a cofactor like ATP, which provides the necessary energy for the enzyme. The efficiency of this reaction is heavily influenced by the relative amounts of the two components. Researchers typically aim for an insert-to-vector ratio that favors the formation of the desired recombinant plasmid.
While sticky ends are preferred because the complementary overhangs stabilize the fragments and make ligation highly efficient, blunt-end ligation is less efficient and requires higher concentrations of ligase and DNA. The reaction is often incubated at a cool temperature, such as 16°C, for several hours or even overnight, as this temperature is a compromise that allows the sticky ends to anneal while the ligase enzyme remains active. The successful completion of this step yields a recombinant plasmid containing the gene of interest.
Introducing the Recombinant Plasmid into Bacteria
The newly created recombinant plasmid must be introduced into a living host organism so it can be replicated, a process called transformation, which is typically performed using Escherichia coli bacteria. Since bacteria do not naturally absorb foreign DNA, they must first be made “competent” to take up the plasmid. This involves treating the cells to temporarily increase the permeability of their cell membranes.
Two main laboratory methods are used to create competent cells and facilitate transformation. Chemical transformation involves treating the cells with calcium chloride or other salts while keeping them on ice. The cells are then subjected to a brief, rapid increase in temperature, known as a heat shock, which causes temporary pores, allowing the negatively charged DNA to enter.
The second method, electroporation, is more efficient and involves subjecting the mixture of competent cells and plasmid DNA to a short pulse of a high-voltage electric field. This electrical pulse creates transient pores in the bacterial cell membrane, through which the DNA is pulled into the cell. Only a small fraction of the bacterial population successfully takes up a plasmid, which makes the next step of identifying the successful cells necessary.
Identifying Successful Clones
This begins with selection, where the transformed bacteria are grown on an agar plate containing an antibiotic. Plasmids are engineered to carry an antibiotic resistance gene, which serves as a selectable marker. Only bacteria that have successfully taken up any plasmid—recombinant or not—will survive and grow into colonies, while the untransformed cells will be killed by the antibiotic.
After initial selection, a screening step is required to identify which of the surviving colonies contain a plasmid with the gene insert. Blue/white screening uses the lacZ gene on the plasmid, which encodes the enzyme beta-galactosidase. The cloning site is located within this gene; if the gene insert is successfully incorporated, it disrupts the lacZ gene, preventing the production of functional enzyme.
The agar plates for this screening contain a compound called X-gal, which turns blue when cleaved by functional beta-galactosidase. Colonies with a non-recombinant plasmid appear blue, while colonies containing the desired recombinant plasmid appear white. Researchers then pick the white colonies for further verification.
A small sample of plasmid DNA is isolated from the candidate colonies. This isolated DNA is then analyzed using techniques like diagnostic restriction digests, which cut the plasmid at specific sites to confirm the size of the inserted fragment. Sanger sequencing determines the exact nucleotide sequence of the insert and its boundaries. This ensures the correct gene was incorporated and is oriented properly within the plasmid.