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Can anyone offer any insight why in SMART-seq2 the RT and TSO primers are designed in a way that cDNA is amplified by semi-suppressive PCR (suggesting that without it, there would be a lot of primer-dimer products amplified) whereas in SMART-seq3/FLASH-seq, the RT/TSO primers carry different anchor sequences for cDNA amplification which does not rely anymore on semi-suppressive nature. So is the primer-dimer formation suppressed by either different RT enzyme usage or reaction buffer conditions in SS3/FLASH-seq or somehow these new RT/TSO primers do not form primer-dimers as efficiently as those in SMART-seq2? Is there then any RT/TSO design principle that one should follow to avoid excess formation of primer-dimers during template-switching RT? Thanks a lot!
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Hi @dasiSo I was discussing this with our VP of tech dev. The product we have on the market at http://rnaconnect.com is doing semi suppressive PCR. (They cDNA Synthesis & Amp Kit uses same PCR primer for both ends). The idea of the same handles on the ends of the PCR product actually has nothing to do with primer dimers. The idea is to suppress overamplification of the small cDNA stuff so that you give the big stuff a chance. It was patented by Clontech but it’s long expired by now. In his opinion and from experience with PacBio, there’s not a huge effect from the handles being the same, partially due to AMPure bead purification being commonplace to save the day and get rid of small stuff? From an informatics stand point, having the same primers on both ends is a pain when trying to figure out the 5' end from the 3' end and this is likely why the field has moved away from this.
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