Pathology, often referred to as the science of diseases, occupies a pivotal role in the…
Polymerase Chain Reaction (PCR) and Quantitative Polymerase Chain Reaction (qPCR) are two molecular biology techniques for amplifying and detecting DNA. While both methods rely on the principle of amplification via DNA polymerase, there are several significant differences between them.
What is PCR?
Polymerase chain reaction (PCR) is a technique for replicating a specific section of DNA multiple times. This process begins with a small amount of template DNA, which is then copied by polymerases, which are enzymes. The number of copies made can be increased by repeating the heating and cooling cycle, which separates the strands of DNA and allows them to be copied again. It is a highly sensitive and specific technique.
PCR produces a large number of identical copies of your target DNA sequence. This product can then be used for additional analysis such as sequencing. When using PCR assays, contamination of source DNA, which can occur when amplified DNA molecules are “carried over” from one round of reactions to the next, can be a problem. Endpoint PCR assays may also be overly sensitive in some cases, because certain bacterial species (such as bacteria in the gut) can be beneficial at low levels (in which case, qPCR may be a better option).
What is qPCR?
qPCR is a technique used to quantify the amount of DNA in a sample. It is a variation of PCR that incorporates fluorescent probes that allow for the detection and quantification of the amplified DNA in real-time (hence why it is also known as real-time PCR). This subsequently allows for a quicker detection of target DNA sequences, reduced cross-contamination risk and enhanced testing sensitivity.
qPCR works by amplifying target DNA to levels that produce a detectable signal. A small amount of a sample (e.g., saliva or urine) is mixed with the building blocks required to replicate the DNA sequence. The presence of fluorescent probes, or DNA binding dyes, in the reaction mixture distinguishes qPCR from older methods. If the sample contains the target DNA sequence (for example, Staphylococcus DNA), the binding dyes will bind, activate, and produce a detectable signal. The photocopied or ‘amplified’ DNA is monitored using fluorescence, which measures light from probes specific to the target DNA of interest. The findings determine whether or not a molecular target exists (e.g., pathogen). In the case of a virus-abundant sample, the measured signal would rise rapidly over time. However, if there is no or very little DNA in the original patient sample, no signal is detected, indicating a negative result.
qPCR is a highly sensitive and specific technique that allows for the quantification of DNA from a small amount of starting material. It is widely used in research and clinical settings for applications such as gene expression analysis, pathogen detection, and diagnosis of genetic disorders. qPCR has several advantages over traditional PCR, including greater accuracy and precision, higher sensitivity, and the ability to detect and quantify multiple targets in a single reaction.
In summary, PCR and qPCR are two powerful techniques used in molecular biology for amplifying and detecting DNA. The ability to accurately and precisely quantify DNA from a small amount of starting material makes qPCR an essential technique in molecular biology.
To learn more about our qPCR solutions, please contact us. We offer the largest menu of qPCR kits in the world through our partner, YouSeq, as well as the option for custom solutions.