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UMI design on SMART template switch oligo in Turchaninova

In the Turchaninova et al protocol, the UMI in the SMART oligo is designed as …UNNNNUNNNNUNNNNU...(rG)4. What is the reason for the interspersed uracils?

Additional questions:

  • The SMART primer contains both Ts and Us. What’s the story there?
  • I’m assuming all the nucleotides are deoxynucleotides (including the Us) except for the final 4 riboGs. Is this correct?


The purpose of uracils is to allow the elimination of adapter molecules using the Uracil-DNA Glycosylase (UDG) enzyme, to ensure they will not interfere with the subsequent PCR reaction. All nucleotides are deoxynucleotides indeed.

PS. If have more questions about the protocol/need some troubleshooting I can forward you to the first author, just write me at my username @ gmail.

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Ha, that’s what I get for just looking at the pictures. (Sorry bout that)

I would like to follow the protocol in this paper to sequence immunoglobulin. I’m testing the method as described in the paper at the moment. But I’m new in this area, I have also a couple of questions:

  1. I don’t really understand the purpose of riboG in the SMART oligo. Will G function the same or it has to be a riboG?

  2. The “Template-free added cytosines” during the cDNA synthesis, I guess this step is crucial for the incorporating UMI to cDNA. Does the addition of CCCC only happen to a certain reverse transcriptase or to any rereverse transcriptase?

  3. Considering the diversity of the VH, I assume that the PCR products after the first PCR (seminested) should be a smear on gel, but instead of a smear, I got several bands of different size. I wonder if you have experienced the same or it has gone wrong in my procedure somewhere?

I would really like to have this method working, but so far it seemed difficult

Appreciate your help