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Old 01-14-2018, 06:17 PM   #31
HiroMishima
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Location: Nagasaki, Japan

Join Date: Aug 2009
Posts: 15
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Quote:
Originally Posted by korostin View Post
Hiro,

Maybe I don't understand main Ampliseq feature correctly? Because for my mind, super-multiplex provided by step-out PCR based on universal identical 5'-tail on all primers. So, primer cleavage means 5'-tail cutting. Also, universal and specific parts of every primer separated by uracil. After PCR all products would be like:

5' universal-tail-U-NNNNNNNNNNNN......................... 3'
3' .......................NNNNNNNNNNNN-U-5'universal-tail 5'

[dots mean nothing only for visualisation]
Because classic DNA polymerases can't read uracil and stop synthesis.
If so, uracil DNA glycosylase and endonuclease IV treatment would produce blind ends. If PCR products have no 5' overhangs, uracil DNA glycosylase and endonuclease IV would create nicks only.

Could you explain, please?
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.

Hiro.
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