The Polymerase Chain Reaction (PCR) is a technique used in molecular biology to create millions of copies of a specific DNA segment from a very small starting sample. This amplification process allows researchers to study genetic material in detail. When scientists perform a genetic cloning experiment, they insert a desired piece of DNA into a carrier molecule, typically a bacterial plasmid. After introducing this modified plasmid into bacteria, a rapid and reliable method is needed to check which bacterial colonies successfully incorporated the target DNA. Colony PCR provides this rapid screening capability.
Why Use Colony PCR
Traditional methods for verifying successful cloning involved growing liquid cultures of selected colonies overnight. Following this growth phase, researchers had to perform a plasmid extraction to purify the DNA. The purified plasmid DNA was then analyzed to confirm the presence and size of the inserted DNA fragment. This multi-day process severely limits the number of colonies that can be screened.
Colony PCR bypasses these lengthy purification steps entirely by allowing the direct testing of bacterial cells taken straight from the agar plate. This reduces the screening time from days to a few hours, increasing the efficiency and throughput of cloning projects. Since no costly reagents for DNA purification are needed, this approach is also cost-effective, making it the preferred method for high-volume screening of bacterial transformation results.
Setting Up the Reaction
The reaction requires a master mix solution containing all the chemical components necessary for DNA amplification. A specialized enzyme, typically a thermally stable DNA polymerase like Taq polymerase, is included to synthesize new DNA strands. This polymerase requires a specific buffer solution, which maintains the optimal pH and provides necessary salts, such as magnesium ions.
Deoxynucleotide triphosphates (dNTPs) are the fundamental building blocks that the polymerase incorporates into the growing DNA chain. The specificity of the reaction comes from the primers, synthetic DNA sequences designed to bind to known regions flanking the inserted DNA or within the cloning vector itself. Using a pair of primers—one forward and one reverse—ensures that only the target sequence is amplified.
Once the reaction tubes contain the template (the colony) and the master mix, they are placed into a thermal cycler, a machine that precisely controls temperature cycles. A typical program begins with an initial denaturation step around 95°C for several minutes, which ruptures the bacterial cells and separates the DNA into single strands. This is followed by 25 to 35 cycles of denaturation (e.g., 94°C), annealing (e.g., 50–65°C, where primers bind), and extension (e.g., 72°C, where DNA is synthesized).
Detailed Step-by-Step Protocol
Preparation begins by labeling the bottom of the agar plate with a grid or numbering system corresponding to the PCR tube positions to prevent sample mix-ups. Maintaining sterility is necessary to avoid contamination from environmental bacteria or other DNA sources.
To collect the bacterial template, a sterilized pipette tip is used to gently touch the top of a single, isolated colony on the agar plate. The tip is then immediately submerged into the pre-prepared PCR tube containing the master mix and swirled gently to release the cells into the solution. Using too much cellular material may inhibit the PCR reaction.
A crucial step involves immediately using the same tip to inoculate a new, labeled spot on a fresh agar plate or to streak the colony onto a liquid growth medium. This backup culture ensures that if the subsequent PCR indicates a successful clone, the viable bacteria are available for future large-scale growth and plasmid purification.
After inoculating the tube and the backup plate, the PCR tubes are briefly spun down in a microcentrifuge to ensure all the liquid and cellular material are at the bottom. The tubes are then securely loaded into the thermal cycler block, and the pre-programmed cycling parameters are initiated.
Interpreting the Gel Electrophoresis
The resulting DNA products are visualized using agarose gel electrophoresis, a technique that separates DNA fragments based on their size and charge. The PCR product is mixed with a loading dye and placed into wells in a solidified agarose gel, which is then subjected to an electric current.
Since DNA is negatively charged, the fragments migrate through the gel matrix toward the positive electrode; smaller fragments move faster and farther than larger ones. To accurately determine the size of the amplified DNA fragment, a DNA ladder is run alongside the samples.
A successful cloning event is indicated by the presence of a distinct band in the gel lane that matches the expected size of the cloned insert or amplicon. For instance, if the target insert was 500 base pairs long, a positive result would show a band aligning with the 500 bp marker on the DNA ladder. Colonies that did not successfully incorporate the DNA insert, or those that incorporated a fragment of the wrong size, will typically show no band or a band of a significantly different length.
The absence of any band suggests the bacterium did not contain the plasmid or the PCR failed entirely due to technical issues. Conversely, the presence of two bands may suggest non-specific amplification or that the colony was not isolated and contained two different plasmids.
Addressing Failed Experiments
When gel electrophoresis results show no bands or bands of the wrong size, troubleshooting is required.
Template Issues
One common issue relates to the template DNA. Picking too much colony material can introduce inhibitors that prevent the polymerase from functioning correctly. Conversely, picking too little material may result in an insufficient amount of template DNA to initiate the reaction.
Primer and Temperature Optimization
The design and concentration of the primers are a frequent source of failure. Non-specific binding can lead to unintended products, while low primer concentration results in poor amplification efficiency. Adjusting the annealing temperature enhances specificity; a temperature that is too low can permit primers to bind loosely to non-target sequences, whereas a temperature that is too high prevents the primers from binding at all.
Using Controls
Researchers should always include both positive and negative controls to validate the experiment’s reliability. A positive control, typically a known plasmid containing the insert, confirms that the master mix and thermal cycler are working correctly. A negative control, where water replaces the bacterial template, ensures that the reagents themselves are not contaminated with foreign DNA. Checking the quality and age of the DNA polymerase and dNTPs is also a simple step to rule out reagent degradation.