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UMI design on SMART template switch oligo in Turchaninova

Protocols
#1

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?

#2

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?
#3

Hi!

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.

1 Like
#4

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

#5

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