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Old 06-05-2018, 09:30 PM   #35
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Location: New York

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Originally Posted by HiroMishima View Post
In our protocol, we performed AmpliSeq multiplex PCR using the following materials:
1) original AmpliSeq primers: containing target-specific sequences with uracil nucleobases but not containing universal sequences
2) KAPA2G FAST Multiplex Kits: uracil-tolerant polymerase and regular dNTPs (A, C, G and T)
3) genomic DNA

The uracil DNA glycosylase and endonuclease IV treatment for the amplicons followed by the AMPure XP cleaning obtained blunt-ended uracil-less fragments being ready for Illumina library construction.

I hope that answers your question.

Hi Hiro,

This seems like an interesting approach. Perhaps you could comment on a couple of things to help make me understand the logic behind your approach.

Only the primers contain uracil and in the subsequent amplicons, the opposite strand would be a non-uracil nucleotide. What makes you think the products are blunt after your treatment with UDG & Endo IV? As opposed to being blunted/having the 3' overhangs being chewed away during the first step of the Kapa library prep protocol? I think this is important for those of us trying to put together a homemade protocol since your paper is the first published evidence of getting AmpliSeq on the MiSeq.

Also, what made you decide to go with Endonuclease IV? One of the modifications I've considered for my own protocol is using Endonuclease VIII & UDG together to create 5'-P moieties that allow direct ligation of the amplicons, but I think I would likely need to treat with DNA Pol 1 or T4 DNA Polymerase to actually chew away the 3' overhangs which would be complementary to the AmpliSeq primer sequences and be uracil-less.
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