To solve the three problems mentioned before, we came up with this multiplex PCR strategy called tem-PCR (for target enriched multiplex PCR).
We use at least two pairs of nested primers for each amplification target, so if there is 10 targets we want to amplify together (multiplex), there will be total of 40 primers (4×10). The inside primers for each target have a tag (a short DNA sequence) that is shared by all the target, and it can be recognized by a pair of primers that we call “superprimers”.
The “trick” is that all these target specific nested primers were included in the reaction at very very low concentration, and they are not labeled. Only the superprimers are included at the regular PCR concentration. The reverse superprimer is biotin labeled, and it has a even higher concentration than the forward superprimer, so that most of the PCR products will be single stranded and easy for downstream detection.
Another “trick” is PCR condition. We use cycling condition to dictate which primers get used at certain stage.
The entire protocol has three stages: (1) enrichment stage, (2) tagging stage, and (3) amplification stage.
During the enrichment stage, the nested, target specific primers are used. Since these primers were used at very low concentration, we give extra long (1 min, 30 sec) time for annealing. So they can find their intended target.
The enrichment stage is when we solved the incompatibility problems. With conventional “multiplex” PCR, each target only use one pair of primers, therefore, only one “optimal” condition for that target. By adding another pair of primer outside, four pairing combinations become possible. Therefore, more chances for that target to get amplified at any given condition. For example, the out-out primers may like 55ºC, but the out-in primers may be good at 60ºC.
At the tagging stage, we set the annealing temperature to 72ºC, at this high temperature, only the long 40nt inside primers will work. The intended result is to force all PCR products been “tagged” by the universal “superprimer” tail.
Then, at the last, high concentration “superprimer” amplify targets exponentially and label them at the same time.
With this strategy, we successfully solved all three problems. We say “success” because tem-PCR gives us three unique advantages:
(1) Automation. No post PCR clean-up, or post hybridization washes.
(2) Semi-quantitative. Because all targets in the reaction will be amplified by the same pair of Superprimer, the exponential phase of the amplification for all targets are carried out at the same efficiency. Therefore, the end-point analysis of the co-amplified targets are semi-quantitative.
(3) Sensitive, specific, and highly reproducible. And better yet, a multiplex panel (a group of targets in one assay) is very flexible. We can add or remove targets, or regrouping targets, without major impact on existing panels.
The tem-PCR method was first published in the Journal of Clinical Microbiology (Han et al. 2006), the same year, Qiagen acquired our previous company, Genaco, and became the owner of that IP. After left Qiagen and while working for HudsonAlpha Institute for Biotechnology, we have developed a new multiplex PCR technology, called arm-PCR (for amplicon rescued multiplex PCR). The new method was first published in PNAS (Wang et al 2009). In that publication, we applied arm-PCR to amplify rearranged VDJs from sorted peripheral blood lymphocites and then directly sequence the amplicon mixture with the Roche 454 platform. It is with this new core technology, iCubate is now developing a fully integrated solution that allow us to perform multiplex PCR automatically, in a disposable and closed cassette.
Both the tem-PCR and arm-PCR methods are patent pending.
* Disclaimer*
Although the words “Diagnosis” or “Diagnostics” may be used in this blog, our products are NOT FDA approved for such uses. We are developing technologies that may eventually have clinical applications, but for now, all our products are for research use only.