Polymerase Chain Reaction (PCR) is a technique that enables scientists to amplify, or make millions of copies of, a specific DNA segment from a small initial sample. Colony PCR is a specific, streamlined variation of this technique, developed primarily for use in molecular cloning applications. It provides a rapid method for screening numerous bacterial colonies to determine which ones have successfully incorporated a desired piece of foreign DNA.
Colony PCR: A Fast-Track Screening Tool
Colony PCR is employed to accelerate the process of identifying bacterial clones that carry a target gene insert. The fundamental difference between this method and standard PCR lies in the preparation of the template DNA. Traditional PCR requires the template DNA to be isolated and purified from the bacterial culture, a process that can take hours or even days.
Colony PCR bypasses this time-consuming purification step by using the bacterial colony as the direct source of template DNA. A small portion of the colony is sampled and added straight into the reaction mix. The initial high-temperature phase of the PCR cycle serves a dual purpose: it denatures the DNA strands and also lyses (breaks open) the bacterial cells, releasing the plasmid DNA directly into the reaction.
Essential Components of the Reaction Mix
The template DNA is the molecule that will be copied. This template is mixed with short, synthetic DNA sequences called primers, which are designed to bind specifically to the start and end points of the desired DNA region. The primers determine exactly which section of the larger DNA molecule will be amplified.
The reaction uses a heat-stable enzyme, typically Taq DNA Polymerase. The polymerase builds new DNA strands by incorporating deoxynucleotide triphosphates (dNTPs), which are the individual building blocks of DNA. Finally, the reaction requires a specialized buffer solution that maintains the optimal pH and provides the necessary salt concentrations, such as magnesium ions.
Executing the Procedure: From Plate to Thermocycler
The physical procedure begins with colony picking, where a sterile instrument, such as a toothpick or a pipette tip, is used to gently touch and collect a small amount of biomass from an individual colony grown on an agar plate. It is a common practice to use the same sampling instrument to inoculate a corresponding liquid growth medium or patch a new plate before placing the remaining cells into the PCR tube. This ensures that a backup culture of the colony is preserved in case the PCR analysis yields a positive result or the reaction fails.
The sampled cells are then thoroughly resuspended in the prepared master mix containing the polymerase, primers, dNTPs, and buffer. Once the reaction tubes are sealed, they are placed into a thermocycler, a machine programmed to rapidly and repeatedly cycle through three distinct temperature phases.
The first step, denaturation, heats the mixture to a high temperature, typically 94°C to 98°C, which separates the double-stranded DNA template into two single strands. This high heat also performs the initial cell lysis necessary to release the template DNA.
Following denaturation, the temperature is lowered to the annealing phase, usually between 55°C and 65°C, allowing the primers to bind to their complementary sequences on the single-stranded template DNA. The final step is extension, where the temperature is raised to approximately 72°C, the optimal working temperature for Taq polymerase. During extension, the polymerase begins synthesizing a new complementary strand of DNA, starting from the bound primer and incorporating free dNTPs. The thermocycler repeats these three phases for 25 to 35 cycles, exponentially doubling the amount of target DNA with each cycle.
Evaluating Successful Amplification
Once the thermal cycling is complete, the exponential amplification of the target DNA sequence has occurred, but the products are still invisible to the naked eye. To visualize and confirm successful amplification, the resulting DNA mixture is analyzed using gel electrophoresis.
In this process, the PCR products are loaded into wells of a gel matrix, which is then subjected to an electric field. Since DNA molecules possess a negative charge due to their phosphate backbone, they migrate through the gel toward the positive electrode. The gel matrix acts like a sieve, separating the DNA fragments based on their size; shorter fragments travel farther and faster than longer ones.
A DNA size ladder, consisting of fragments of known lengths, is run alongside the samples to provide a reference scale. Successful amplification is confirmed by the appearance of a distinct, visible band on the gel that corresponds precisely to the expected size of the gene insert. The absence of a band, or the presence of a band at an incorrect size, indicates that the bacterial colony did not contain the desired insert or that the reaction failed.
Validation of results is further supported by the use of controls run simultaneously with the samples. A positive control, using a template known to yield amplification, confirms that the reaction components and conditions were functional. Conversely, a negative control, which typically contains all components except the template DNA, is run to ensure that the reagents themselves are not contaminated with extraneous DNA. These controls are important for interpreting the results accurately and confirming that the observed bands are genuinely the product of the target colony.