Singleplex PCR uses one pair of primers to amplify a single target. Multiplex PCR uses several pairs of primers to amplify more than one target sequence. Both are performed in a single reaction tube. Multiplex PCR is often the preferred option due to decreased reaction time and efficient use of both starting material and reagents.
PCR Multiplexing Methods
Optical Channel Multiplexing
The most common way of performing PCR multiplexing is through the use of various optical channels. Separate probes specific to each target sequence are each labeled with a unique combination of fluorophores. The use of these unique fluorophores allows different target sequences to be detected in separate optical channels.
The number of optical channels needed depends on the number of different target sequences to be detected within the reaction. Instruments supporting 2-4 optical channels are commonly available.
Optical channel multiplexing is only compatible with an assay utilizing probe detection chemistry. It is not suitable with a dye-based detection assay, due to the non-specific product amplification by the DNA binding dye. The dye intercalates with all double-stranded DNA, making distinction between the fluorescent signals of different target sequences impossible.
Screening assays use multiple target-specific primers, but detect the fluorescence emitted from amplicons through the use of only one optical channel. This means that all of the target sequences, if present in the sample, will produce fluorescent signals that will be detected within that same single optical channel.
The results do not distinguish between different amplified products. However, screening assays save time and reduce costs since only the samples that have been flagged as positive for a target sequence will undergo further analysis. These assays are compatible with either intercalating dye or probe detection chemistries.
Screening assays are typically used in situations where there are many samples to be tested, each for the presence of multiple targets, such as a food/beverage quality control assay that screens for multiple pathogens.
While melt curve analysis (MCA) is typically used to detect non-specific product amplification, it may also be used for multiplexing.
MCA is applied to the identification of several specific PCR products by measuring the decrease in fluorescence intensity as the double-stranded DNA dissociates into single-stranded DNA. The temperature at which 50% of the total double-stranded DNA present separates is referred to as melting temperature (Tm). The assay is designed such that every amplicon has a different melting temperature. Tm multiplexing may then be achieved by differentiating PCR products using the varying melting temperatures of the different amplicons.
Using Tm multiplexing, the MCA data below shows the differentiation of GAPDH and HPRT amplicons, with each peak corresponding to a specific amplicon.
Melt Curve Analysis of HPRT & GAPDH Reaction Products via Eva Green Assay
Singleplex PCR Advantages + Disadvantages
+ Ease of Design
With a single target assay, there is no competition for reaction components, making assay design much easier.
+ Decreased Instrument Cost
Only a single channel real-time PCR instrument is required for singleplex assays.
+ Choice of Detection Chemistry
Singleplex assays may be performed using either a dye or probe detection chemistry, affording the user additional control over assay specifications.
- Inefficient for Multiple Target Assays
Using several singleplex reactions to perform a multiple target sequence assay means an increase in reagent quantity consumption and sample material requirements. Additional costs and labor are incurred as a result.
Multiplex PCR Advantages + Disadvantages
+ Maximum Efficiency
Using multiplex PCR rather than several singleplex PCR reactions maximizes the use of limited starting material and lowers reagent costs for assays requiring amplification of several target sequences.
+ Internal Control Option
An internal control coupled with the sample confirms the absence of inhibitors in the reaction. Internal controls may also serve to verify that the extraction process was complete.
+ SNP Genotyping via Allelic Discrimination Plot
A multiplexed end-point assay for detecting variants of a single nucleotide sequence would classify unknown samples as either homozygous or heterozygous and would show up as clusters in the allelic discrimination plot.
+ Useful for High-Volume Environments
Multiplex assays are excellent for labs needing to perform a large quantity of the same test repeatedly.
A non-optimized multiplex assay may experience primer-primer hybridization or incorrect primer-template binding. This produces inaccurate Cq (quantification cycle) values.
- Multiple Optimization Rounds
Competition for reaction components causes low-abundance targets to be outcompeted by high-abundance targets. Multiple rounds of optimization are often needed to set the appropriate primer concentration.