The Polymerase Chain Reaction (PCR) and restriction enzymes are both fundamental tools in molecular biology. Restriction enzymes are not used directly within the Polymerase Chain Reaction (PCR) itself. PCR amplifies specific DNA sequences, while restriction enzymes cut DNA at precise locations. These two distinct processes are frequently used sequentially in broader molecular biology workflows.
How PCR Works
PCR is a laboratory technique that creates millions of copies of a specific DNA segment from a small initial amount. The process relies on a DNA template, primers (short synthetic DNA molecules), DNA polymerase (like Taq polymerase), and nucleotides (A, T, C, G).
Amplification occurs through a cyclical process involving three main steps. First, denaturation heats the mixture to 94-98°C to separate double-stranded DNA into single strands.
Next, during annealing, the temperature lowers to 50-65°C, allowing primers to bind to complementary sequences. Finally, in the extension step, the temperature rises to approximately 72°C, the optimal temperature for Taq polymerase, which synthesizes new DNA strands by adding nucleotides. These steps repeat 25-40 times, leading to an exponential increase in the target DNA sequence.
How Restriction Enzymes Work
Restriction enzymes, also known as restriction endonucleases, are proteins found in bacteria. They serve as a defense mechanism, protecting bacteria from invading viruses by recognizing and cutting foreign DNA.
Each restriction enzyme identifies a specific, short sequence of nucleotides on the DNA molecule, called a recognition site. Once located, the enzyme makes precise cuts within or near that sequence, cleaving the DNA double helix.
The cuts can result in either “sticky ends” (short single-stranded overhangs) or “blunt ends” (where the DNA is cut straight across both strands). Their ability to cut DNA at predictable sites makes them useful tools in genetic engineering for tasks like gene cloning and DNA mapping. Most restriction enzymes function optimally around 37°C.
Why They Are Not Used Together in PCR
Restriction enzymes are not used directly within the PCR reaction due to conflicting functions and operational requirements. PCR aims to synthesize and amplify DNA, while restriction enzymes cut and fragment DNA. If active during PCR, they would continuously cleave the DNA template and newly synthesized products, preventing amplification.
Temperature requirements also present an incompatibility. PCR cycles involve high denaturation temperatures (94-98°C) to separate DNA strands. Most restriction enzymes are sensitive to heat and would be permanently denatured and inactivated at such temperatures. Conversely, their optimal activity is around 37°C, too low for PCR’s denaturation and extension steps.
When They Are Used in Tandem
While not combined in a single reaction, PCR and restriction enzymes are frequently employed sequentially in various molecular biology applications. One common application is gene cloning, where PCR amplifies a specific gene or DNA fragment. Following amplification, restriction enzymes cut the PCR product and a recipient plasmid vector, creating compatible ends for gene insertion. PCR can also incorporate specific restriction enzyme recognition sites into amplified DNA, facilitating subsequent cutting and ligation for cloning.
Another tandem use is in Restriction Fragment Length Polymorphism (RFLP) analysis. Here, PCR amplifies a DNA region that may contain variations. Restriction enzymes then digest the amplified DNA. Differences in the DNA sequence, such as single nucleotide changes, can alter or create new restriction sites, leading to different-sized DNA fragments after digestion. This helps identify genetic variations among individuals. Restriction enzymes are also used in advanced PCR methods like droplet digital PCR, where they fragment large DNA genomes into smaller pieces to improve droplet generation efficiency and accurate quantification.