The Polymerase Chain Reaction (PCR) is a widely used molecular biology technique. It creates millions of copies of a specific DNA segment from a tiny starting amount. This amplification allows scientists to study DNA, identify pathogens, or analyze genetic material. Successful PCR results depend on controlling numerous elements within the reaction.
Key Ingredients in the Reaction Mix
DNA polymerase is an enzyme responsible for synthesizing new DNA strands. Thermostable versions, like Taq polymerase, are commonly used because they withstand the high temperatures of the PCR cycle. Enzyme concentration influences efficiency; too little leads to incomplete amplification, while excessive amounts increase non-specific binding.
Magnesium ions (Mg2+) serve as a cofactor for DNA polymerase, necessary for the enzyme to function correctly. They impact polymerase activity and primer binding specificity. An optimal Mg2+ concentration, typically 1.5 mM to 2.5 mM, promotes efficient target DNA amplification. High concentrations can lead to non-specific binding and unwanted products. Low concentrations reduce polymerase activity, resulting in low or no yield.
Deoxynucleotide triphosphates, or dNTPs, are the fundamental building blocks for new DNA strands. These include dATP, dGTP, dCTP, and dTTP, which must be in balanced concentrations. Each dNTP is supplied at 0.2 mM to 0.4 mM. An imbalance or insufficient amount of any dNTP limits new DNA synthesis, reducing the PCR yield.
The PCR buffer system maintains the optimal chemical environment, stabilizing pH. Buffers contain salts and detergents that ensure DNA polymerase activity and predictable DNA strand behavior. Different DNA polymerases perform best in varied buffer compositions, requiring a compatible buffer for the chosen enzyme. Maintaining a stable pH, usually 8.3 to 8.8, is important for enzyme activity throughout PCR temperature fluctuations.
The Role of DNA and Primers
The DNA template must have high purity and integrity. Contamination or degradation can lead to inaccurate results or reaction failure. Template DNA quantity must be appropriate; too little may result in no amplification, while high amounts can hinder the reaction or lead to non-specific products.
Primers are short, synthetic DNA strands designed to bind to specific regions of the target DNA. Design considers length, sequence, and melting temperature (Tm). Primers are 18 to 24 nucleotides long, with a Tm of 55°C to 65°C, ensuring stable and specific binding. Poorly designed primers may bind to multiple locations, amplifying unintended fragments, or fail to bind, resulting in no amplification.
Primer sequence determines attachment to the template DNA. Primers should have a balanced guanine (G) and cytosine (C) content, 40-60%, because G-C pairs form stronger bonds than adenine (A)-thymine (T) pairs. This balance helps ensure consistent binding strength. Primer-dimer formation, where primers bind to each other, can occur if primers have complementary sequences, reducing primer availability for the reaction.
Primer concentration influences amplification efficiency and specificity. Each primer is typically 0.1 µM to 0.5 µM. Low primer concentration can lead to inefficient reactions and low product yield. High concentrations promote non-specific binding, increase primer-dimer formation, and reduce correct product yield by competing for components.
Optimizing the PCR Machine’s Program
The thermal cycler controls temperature changes for amplification. The program involves three main steps, each with specific temperature and time settings optimized for DNA synthesis. Denaturation, the first step, heats the reaction to 94°C to 98°C for 15 to 30 seconds. This separates double-stranded DNA into single strands, accessible for primer binding.
Annealing follows denaturation, lowering the temperature for primers to bind to complementary single-stranded DNA. The annealing temperature is usually 3-5°C below the primers’ Tm, often 50°C to 65°C, lasting 15 to 30 seconds. Correct annealing temperature is important; too high leads to inefficient primer binding and low yield, while too low causes non-specific binding and unwanted fragments.
Extension, the final step, raises the temperature to the DNA polymerase’s optimal activity range, typically 68°C to 72°C. This phase, usually 30 seconds to several minutes depending on target length, allows the polymerase to synthesize new DNA strands by adding dNTPs from the primer binding site. Extension time should be sufficient for polymerase to complete target DNA synthesis. These three steps repeat for multiple cycles, leading to exponential target DNA amplification.
The number of amplification cycles influences final product amount and potential for errors. PCR reactions run for 25 to 40 cycles. More cycles produce more amplified DNA, beneficial with small template amounts. However, too many cycles increase non-specific products and amplify minor contaminants. Prolonged cycling can exhaust components and introduce more errors.
Addressing Unwanted Interferences
Substances in a sample can negatively affect PCR by acting as inhibitors. Inhibitors interfere with amplification stages, leading to reduced efficiency or complete failure. Common inhibitors include proteins, polysaccharides, heme from blood, or humic acids from soil, often introduced during DNA extraction. They can bind directly to DNA polymerase, inactivating it, or interfere with primer binding.
Other inhibitors, like detergents (SDS), phenol, or salts, can be carried over from DNA purification. These chemicals disrupt enzyme structure or alter the ionic environment for optimal polymerase activity. Some inhibitors can chelate magnesium ions, making them unavailable for DNA polymerase. Their presence can reduce PCR sensitivity, meaning target DNA may not be successfully amplified.
DNA sample source dictates the type and concentration of potential inhibitors. Blood samples may contain heme and immunoglobulin G, both PCR inhibitors. Plant extracts can contain polysaccharides and polyphenols, while forensic samples from soil or ancient remains often contain humic substances. Effective DNA purification methods are important to remove these inhibitors, ensuring extracted DNA is suitable for PCR. Even small amounts of inhibitors can impact the reaction’s outcome.