First used as a method in detecting deletions in the dystrophin gene back in 1988, multiplex PCR has since made its way into many protocols across a variety of research areas. While still used in gene deletion analyses, multiplex PCR, as well as qPCR, is an incredibly useful tool in many applications, such as SNP genotyping, pathogen detection, and food analysis to name just a few.
Multiplex allows for the researcher to detect multiple targets in a single reaction well. The prerequisite for this is using more than one primer pair for simultaneous target amplification and analysis.
Here are some of the key benefits you will see when adopting multiplexing into your workflow:
Saves money
Saves rare samples
More reliable results
Multiplex PCR is a useful tool in any laboratory working with repeating samples and targets. Incorporating it into your workflow allows you to work through your samples faster while using fewer samples as well as consumables. Multiplexing can be applied both in endpoint PCR as well as in real-time qPCR.
In endpoint PCR, gene targets are discriminated by amplicon size and detected typically via gel electrophoresis. The amplicon size may be limited by the properties of a DNA polymerase. Typical Taq DNA polymerases allow amplification of up to 5 kb fragments. With endpoint PCR generally, more targets can be detected in one reaction compared to real-time qPCR. In qPCR, the amplicon length is rather limited, typically between 100-200 bp, and the amplicons are detected in real-time using mostly target-specific hydrolysis probes (i.e TaqMan) or dsDNA intercalating dyes (i.e. SYBR® Green, EvaGreen®, or other similar dyes). Multiplex qPCR with hydrolysis probes is highly adopted in the diagnostic sector. It is used for detecting several pathogens and internal control in the same sample in an extremely specific manner. Its time-saving, cost-efficient, and increased reliability features make it highly beneficial for routine and high throughput experiments.
Each target gene must bind a differently labeled probe to set its amplification signal apart from the other targets in the same reaction, as each label emits fluorescent light at a different wavelength that is detected by specific channels in the qPCR cycler.
When starting your next project, consider leveraging the benefits of multiplexing to increase your efficiency!
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There are two options for target detection when doing qPCR - either you use dye or probe-based detection. Today we are going to take a deep dive into the world of dye-based qPCR, which was invented during the early nineties by Russell Higuchi [1] and is still used daily around the world.
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