Amplifying Plasmid DNA Using Polymerase Chain Reaction (PCR)

Scientists often need to make many copies of a specific piece of DNA for applications ranging from genetic research to medical diagnostics. Two components used in this process are plasmids and the polymerase chain reaction (PCR). Plasmids are DNA carriers, and PCR is a technique for copying a specific DNA sequence. Understanding how these tools work together provides insight into how scientists study and manipulate genetic material.

What Are Plasmids?

Plasmids are small, circular DNA molecules found in bacteria, separate from the main chromosomal DNA. They can replicate independently within a host cell because they contain an origin of replication, a sequence that the host cell’s machinery uses to start DNA copying.

These features make plasmids adaptable laboratory vectors for carrying foreign DNA. To identify cells that have taken up the plasmid, vectors are engineered with selectable markers. A common marker is an antibiotic resistance gene, which allows only bacteria with the plasmid to grow when the antibiotic is present.

Many lab-use plasmids also include a multiple cloning site (MCS). The MCS is a short DNA segment with multiple restriction sites, which are specific sequences cut by enzymes. This provides a location for researchers to insert a DNA fragment into the plasmid.

Principles of Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a method used to amplify a specific DNA segment. The technique creates many copies from a small starting amount. The process uses temperature cycles and requires a DNA template, primers, a heat-stable DNA polymerase, and nucleotide building blocks. Primers are short DNA sequences designed to match the start and end of the target region, ensuring only that segment is amplified.

PCR involves a cycle of three main steps that are repeated multiple times.

• Denaturation: The mixture is heated to 94-98°C, breaking the hydrogen bonds of the DNA and separating it into two single strands.
• Annealing: The temperature is lowered to 50-65°C, allowing the primers to bind to their complementary sequences on the single-stranded DNA.
• Extension: At around 72°C, a heat-stable DNA polymerase (like Taq polymerase) attaches to the primers. The enzyme then synthesizes a new DNA strand complementary to the template, creating a new double-stranded DNA molecule.

This three-step cycle is repeated 20 to 40 times, with each cycle doubling the amount of the target DNA, leading to an exponential increase in the number of copies.

Amplifying Plasmid DNA Using PCR

PCR is frequently applied to plasmids, using the plasmid itself as the DNA template. Primers can be designed to target and amplify a specific region, such as an inserted gene, allowing for its selective copying.

A common use of PCR with plasmids is to verify the presence and size of a DNA insert. Using primers that bind to regions flanking the insertion site will generate a product of a predictable size if the insert is present. Analyzing the product’s length confirms the successful construction of the recombinant plasmid.

PCR is also a tool for modifying DNA sequences within a plasmid. Primers can be designed to add sequences, such as restriction enzyme sites, to the ends of the amplified DNA. This allows the fragment to be easily inserted into a different plasmid for subsequent cloning.

Another application is the linearization of a circular plasmid. Some experiments require a linear (straight) form of the plasmid DNA. By designing primers that bind back-to-back, PCR can amplify the entire plasmid, converting it into a linear piece of DNA. This technique prepares the DNA for specific types of cloning or manipulation.

Uses of Plasmid DNA PCR

A primary use is in gene cloning, where PCR generates large quantities of a specific gene from a plasmid template. The amplified DNA can be inserted into expression systems designed to produce the protein encoded by the gene. This is a step for studying gene function and protein characteristics.

Another application is the rapid screening of bacterial colonies, known as colony PCR. After introducing a plasmid into bacteria, scientists must identify which colonies contain the correct DNA insert. A small amount of a colony can be used directly as the PCR template, which quickly reveals if the desired plasmid is present and saves significant time.

The technique is also used for site-directed mutagenesis, a process for making specific changes to a DNA sequence. Using a plasmid template, primers with a slight mismatch to the original sequence are used. PCR incorporates this mismatch into the new DNA, creating a modified gene. This allows researchers to study how specific changes in a gene’s code affect its function.