Both PCR and qPCR are polymerase chain reaction techniques used to amplify specific sections of DNA. The main difference between the two is that qPCR is a real-time method, while PCR is not.
This means that with qPCR, you can monitor the amplification of your target DNA in real-time as it is happening. This guide will introduce you to both methods and explain the main differences between them.
What is PCR?
PCR – Polymerase Chain Reaction – is a lab technique that was developed by Kary Mullis in the 1980s, and today is used to create a large amount of DNA from a small amount of DNA.
PCR is usually a step used in other processes, for example, DNA sequencing, detection of pathogens, and gel electrophoresis. PCR is an essential diagnostic technique which is frequently used in fields such as virology, microbiology, parasitology, mycology and dentistry.
Polymerase chain reaction (PCR) is a method used to make multiple copies of a specific section of DNA. This process starts with a small amount of template DNA, which is then copied by enzymes called polymerases. The number of copies made can be increased by repeating the cycle of heating and cooling, which separates the strands of DNA so that they can be copied again.
The final product of PCR is a large number of identical copies of your target DNA sequence. This product can then be used for further analysis, such as sequencing or Southern blotting. PCR can also be used to make cDNA from mRNA template molecules.
What is qPCR (Real-Time PCR)?
Quantitative polymerase chain reaction (qPCR or quantitative PCR) is very similar to PCR, but it also allows you to quantify the amount of target DNA present in your sample. qPCR involves adding fluorescent dyes or probes that bind to your target DNA during amplification. These probes emit light at different wavelengths when they are excited by a laser.
By measuring the light emitted at each wavelength, you can determine how much target DNA was present in your sample before amplification began. qPCR can also be used to monitor the progress of amplification in real time by measuring the increase in fluorescence over time.
What is Reverse-transcription PCR?
Reverse-transcription PCR (RT-PCR) is a type of PCR that is used to make cDNA from mRNA template molecules. RT-PCR is typically used to detect and quantify RNA viruses, such as HIV and hepatitis C.
To carry out RT-PCR, you first need to reverse transcribe your mRNA template into cDNA using reverse transcriptase enzymes. You can then use PCR to amplify your target cDNA sequence. RT-PCR is a very sensitive method, and it can be used to detect extremely low levels of RNA viruses in a sample.
qPCR comes into its own with the ability to measure gene expression. In gene expression, mRNA is synthesised when a section of DNA is read, the mRNA is then used to produce proteins.
In the process of reverse transcription, reverse transcriptase can be used to create complementary DNA (cDNA) which will be copies of the RNA being studied. qPCR is then carried out on the cDNA to give accurate measurements of the amount produced.
RNA can be used in both PCR and qPCR, if RNA is used in PCR it is known as RT-PCR, if RNA is used in qPCR it is known as qRT-PCR.
Differences between PCR and qPCR
The main difference between PCR and qPCR is that qPCR is a real-time method, while PCR is not. This means that with qPCR, you can monitor the amplification of your target DNA in real-time as it is happening.
Another difference between the two methods is that qPCR requires the use of fluorescent dyes or probes, while PCR does not. These probes allow you to quantify the amount of target DNA present in your sample.
- PCR is typically used to amplify DNA for sequencing or other downstream applications.
- qPCR is typically used to detect and quantify RNA viruses.
- RT-PCR is a type of PCR that is used to make cDNA from mRNA template molecules.
Applications and uses of PCR and qPCR
PCR is a relatively simple process which will detect the presence or absence of DNA. PCR is used in many different fields, including forensics, medicine, and biotechnology. It is frequently used to amplify small pieces of DNA for sequencing or other downstream applications.
PCR can also be used to make cDNA from mRNA template molecules.
qPCR gives real-time data measuring reaction rates providing information about the quantity of DNA present in the sample at any given time, essentially, it is capable of measuring the rate of gene expression.
In molecular biology, this facilitates an understanding of protein synthesis and therefore biological pathways in health and disease. Real-Time or qPCR is useful for the detection of gene expression, presence and amount of pathogens and RNA as well as for validation of DNA microarray results and for environmental applications.
qPCR has the advantage of high throughput and a broader dynamic range as it can detect wide-ranging levels of expression within the same well. Because of its multiplexing capabilities where multiple targets can be amplified within the same experiment, high throughput and rapid results, it can be more economical to run.
RT-PCR is a type of PCR that is used to make cDNA from mRNA template molecules. RT-PCR is typically used to detect and quantify RNA viruses, such as HIV and hepatitis C.
Both PCR and qPCR are essential methods with many different applications.
Equipment for use with PCR and qPCR
There are many different types of equipment that can be used for PCR and qPCR. The most common type of equipment used is a thermal cycler, which is used to heat and cool your sample to allow DNA amplification to occur.
Other common types of equipment include reverse transcriptase enzymes, fluorescent dyes or probes, and lasers. Depending on your specific application, you may need other types of equipment as well.
PCR and qPCR are essential methods used in many different fields. By understanding the main differences between them, you can choose the best method for your specific application.
In conclusion, PCR and qPCR are both methods used to amplify specific sections of DNA.
However, qPCR is a real-time method that allows you to monitor the amplification of your target DNA as it happens, while PCR does not. Ultimately, both methods can be used to generate multiple copies of your target DNA sequence for further analysis.
qPCR is the best choice for the amplification, characterization, quantification and analysis of nucleic acids. Used with fluorescent dyes, It is perfect for use with optical technology, therefore good quality, clear microplates and films must be used. View our qPCR product range.
PCR is also used to amplify and detect short sequences of DNA for subsequent analysis e.g. gel electrophoresis. Microplates and films do not need to be clear as PCR is not used in optical applications.
It is, however, advisable to choose good quality microplates that will be reliable and durable in the long term.
For further advice and support, the iST team is available for you, please contact us for information.