Polymerase Chain Reaction (PCR) is a technique used to amplify a specific DNA sequence, creating millions of copies from a small sample. Mutagenic PCR is a variation of this method designed to intentionally introduce targeted changes, or mutations, into the DNA being copied. The goal is to create an altered version of a gene. By changing the genetic blueprint, researchers can investigate the relationship between a gene’s sequence and its function.
Applications of Induced Mutations
A primary use of induced mutations is to study the function of genes and the proteins they encode. By altering a single amino acid in a protein’s sequence, researchers can observe how that change affects the protein’s properties and function. This systematic alteration allows scientists to map the functional regions of a protein, determining which parts are responsible for its activity.
This technique is also used in protein engineering to improve proteins for industrial or therapeutic purposes. For example, an enzyme can be modified to become more efficient, withstand higher temperatures, or act on a different target molecule. These engineered proteins have applications in biofuel production and medical treatments.
Induced mutations are also used to model human genetic diseases. Researchers recreate the specific genetic mutations found in patients with inherited disorders. This allows for a detailed study of the molecular mechanisms that cause the disease and provides a platform for testing new drugs and therapies.
Methods for Introducing Mutations
Overlap extension PCR is a common technique that uses two stages. In the first stage, two separate PCR reactions are run. Each reaction uses a standard outer primer and a custom mutagenic primer containing the desired DNA change. These reactions produce two DNA fragments that carry the mutation and have overlapping sequences at one end.
In the second stage, these two fragments are combined. The overlapping ends bind together, and a DNA polymerase extends them to create a single, full-length DNA product. This final product contains the targeted mutation integrated into its sequence.
An alternative is the megaprimer method, which also involves two PCR rounds. The first reaction uses a mutagenic primer to create a large, single-stranded DNA fragment containing the mutation, known as a “megaprimer.” This megaprimer is then used in a second PCR to synthesize the entire target plasmid, incorporating the mutation.
Essential Components and Primer Design
Mutagenic PCR requires several components, with the mutagenic primers being the most important. These are short, synthetic DNA strands designed to bind to the template DNA. They are created with a deliberate mismatch in their sequence that corresponds to the desired mutation.
For proper design, the intended mutation is located near the center of the primer. This central mismatch is flanked by 10 to 15 bases that perfectly match the template DNA, ensuring the primer binds securely during the reaction.
A high-fidelity DNA polymerase is also required. Standard polymerases can introduce random errors during amplification, but a high-fidelity polymerase has a proofreading capability to correct these mistakes. This ensures the only mutation in the final product is the one intentionally introduced.
The reaction also requires a high-quality, purified DNA template, which is a circular piece of DNA called a plasmid containing the gene of interest. A pure template is necessary because contaminants can interfere with the PCR reaction.
Confirming the Intended Mutation
After PCR amplification, the product must be verified. This step ensures the intended mutation was correctly introduced and that no unwanted mutations were accidentally created during amplification.
The standard method for this confirmation is DNA sequencing, with Sanger sequencing often used for its high accuracy. The entire gene amplified from the final PCR product is sequenced to determine its precise order of nucleotide bases.
This new sequence is then compared directly to the original, non-mutated sequence. This alignment confirms if the correct change was made and allows a scan for any off-target mutations.