A plasmid is a small, circular piece of double-stranded DNA that exists independently within a bacterial cell, separate from the main bacterial chromosome. Plasmids can replicate on their own and often carry genes, such as those that confer antibiotic resistance. In biotechnology, scientists use molecular tools to cut open a plasmid and insert a gene or DNA sequence from a different source, creating a recombinant plasmid. Because combining DNA from two distinct sources is complex and rarely works perfectly, rigorous checking is necessary at every step.
The Necessity of Screening
The process of creating a recombinant plasmid and getting it into a host cell, known as molecular cloning, is inherently inefficient. The two main steps, ligation and transformation, have low success rates. Ligation, where a DNA enzyme connects the new gene into the opened plasmid, often results in the plasmid closing back on itself without the insert.
Transformation, the process of coaxing a bacterial cell to take up the newly constructed recombinant plasmid, is also highly inefficient. Only a small fraction of host cells successfully incorporate a plasmid, even under optimized conditions. This low efficiency means that most resulting bacterial cells either contain no plasmid or contain the original, non-recombinant plasmid. Therefore, rigorous screening is required to isolate the few successful clones from the failures.
Initial Selection: Identifying Transformed Cells
The first layer of checking is called selection, which identifies only those host cells that have successfully taken up any plasmid. Laboratory plasmids are engineered to contain a selectable marker, usually a gene providing resistance to a specific antibiotic, such as Ampicillin or Kanamycin. This resistance gene is located on the circular plasmid DNA.
When bacterial cells are grown on a selective agar plate containing the corresponding antibiotic, only cells harboring a plasmid can survive and multiply. Cells that failed to take up a plasmid die. This selection step filters out non-transformed cells, allowing only cells with a plasmid to form colonies. This initial selection confirms only the presence of a plasmid and does not confirm if it contains the intended genetic insert.
Detailed Verification of the Insert
After initial selection, a second, more rigorous layer of verification is necessary to confirm that the surviving colonies contain the plasmid with the correct genetic insert. This step ensures the final DNA blueprint is accurate before proceeding to costly or time-consuming downstream applications. Various molecular biology techniques are employed to verify the size, orientation, and sequence of the inserted DNA fragment.
Restriction Enzyme Digest
One common method is the restriction enzyme digest, which involves using specific DNA-cutting enzymes. Scientists choose enzymes that cut the plasmid at known sites flanking the insert, or within the insert itself, to release the inserted DNA fragment. Running the cut DNA on a gel allows researchers to visualize the resulting fragments and confirm the correct insert size and orientation.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) is often used as a quick screen directly on the bacterial colonies. This method uses specific DNA primers that only recognize and amplify the inserted gene sequence. If the PCR yields a product of the expected size, it suggests the insert is likely present in that particular bacterial clone.
DNA Sequencing
The definitive confirmation is DNA sequencing, which reads the exact order of the nucleotide bases in the plasmid. Sequencing is especially important when the inserted DNA was generated by PCR, a process that can sometimes introduce small errors or mutations. This step ensures that the gene’s sequence is flawless, preventing the production of a faulty or non-functional protein.
Consequences of Unverified Plasmids
Failing to properly identify and verify a recombinant plasmid can have significant and costly ramifications across research and industry.
Laboratory Research
Using an incorrect plasmid leads to failed experiments and the loss of valuable research time. Studies suggest that a substantial proportion of lab-made plasmids may contain undetected errors. These errors directly contribute to irreproducible scientific findings.
Industrial Applications
In industrial applications, such as producing therapeutic proteins like insulin or monoclonal antibodies, an unverified plasmid results in financial loss and product failure. If the plasmid lacks the correct genetic code, the manufacturing process will produce the wrong protein or no protein at all. This high-stakes work requires absolute certainty in the genetic template.
Medical Applications
The consequences are most severe in medical applications, such as gene therapy, where plasmids deliver therapeutic genes into a patient’s cells. An incorrect plasmid could introduce the wrong genetic material or fail to produce the necessary therapeutic agent, posing serious health risks. Errors in plasmids used for gene therapy vectors compromise both the efficacy and safety profile of the final product.