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DNA intercalating dyes (e.g., SYBR Green) are not supported due to incompatibility with Countable PCR’s single-molecule imaging and counting workflow. However, if you are currently using DNA intercalating dye-based assays, transitioning to Countable PCR is straightforward with Universal Multiplexing (UM). UM is a unique chemistry that helps you seamlessly adopt your existing assay design to Countable PCR with a simple change to one of the primers. What’s more - you can combine any existing assays designed for intercalating dyes into a multiplexed Countable PCR assay, up to 4-plex without any synthesis of custom probes.
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Universal Multiplex (UM) chemistry simplifies assay design by eliminating the need for gene-specific probes through the incorporation of a UM-specific adapter sequence at the 5’ end of either the forward or the reverse primer. This adapter creates a probe-specific binding site on your amplicon, enabling precise fluorescent detection by UM probes present in a pre-optimized formulation (Universal Multiplex Set A Kit Cat # KT0005) that you include in the Countable PCR reaction. Only standard desalted primers are needed as in DNA intercalating dye-based assays. No expensive and lengthy custom probe synthesis is required. On top of that, you can combine multiple existing 1-plex assays to form a multiplexed assay using the UM chemistry, up to 4 channels (i.e. 4 plex) at a time.
For each assay, append the desired UM adapter sequence (listed below) to the 5' end of one of your two primers. In most cases, you start with the forward primer. The UM adapter sequence should match the choice of UM Probe (UM-1 to -4); each has a specific channel assigned. The primer with the appended UM adapter sequence is referred to as the ‘UM primer’. The other primer, in this case, the reverse primer, remains unchanged and is referred to as the ‘Non-UM primer’.
The resulting UM and Non-UM primers can be ordered from any DNA synthesis provider with standard desalting. If your application requires high specificity, you can order with HPLC purification to remove any non-specific artifacts that may arise from traces of incompletely synthesized oligos.
Finally, it's always best practice to experimentally validate any new assay on the Countable platform. Assays that perform well on other platforms are likely to demonstrate similar efficiency on the Countable platform.
| UM Probe | UM adapter sequences |
|---|---|
| 🔵 UM-1 Probe | 5’-TAGAAGGCACAGTCGAGG-3’ |
| 🟢 UM-2 Probe | 5’-CAGAAGACGGCATACGAGAT-3’ |
| 🟠 UM-3 Probe | 5’-ACCGTAGAGTCCGAGCAA-3’ |
| 🔴 UM-4 Probe | 5’-GAAGCGTTTATGCGGAAGAG-3’ |
In the following example, the existing forward primer has had the UM-1 probe sequence appended to the 5’ end of the sequence. These primers are now ready to be ordered and tested on the Countable platform.
| Assay Type | Forward Primer | Reverse Primer |
|---|---|---|
| DNA intercalating dye-based | 5’-CACCATTGGCAATGAGCGGTTC-3’ | 5’-AGGTCTTTGCGGATGTCCACGT-3’ |
| Universal Multiplex | 5’-TAGAAGGCACAGTCGAGGCACCATTGGCAATGAGCGGTTC-3’ | 5’ - AGGTCTTTGCGGATGTCCACGT-3’ |
The addition of the UM adapter to existing primers may occasionally result in hairpin structures, which may hinder assay performance. To prevent this, use online tools (e.g., IDT's OligoAnalyzer) to check your UM primer. Aim for no hairpin structures above 35 °C. If hairpins are present, try adding the UM probe sequence to your reverse primer and re-evaluate. If problematic hairpins remain, consider selecting a different UM adapter sequence and its assigned channel. The IDT OligoAnalyzer tool can also identify dimers. If primer dimer is predicted, redesigning the assay may improve assay performance.
UM chemistry offers the ability to combine multiple 1-plex assays previously designed for DNA intercalating dyes to form multiplexed Countable PCR assays. However, as the individual 1-plex assays are not previously designed to work as a multiplex, unintended oligo interactions within the multiplex may lead to non-specific amplification. We highly recommend that you confirm the primers (including the added UM adapters) in the desired multiplex assay don't interact with each other through in silico analysis.
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