Hello!
I am trying to sequence (Miseq) a custom amplicon from many samples using the Nextera indexes. For those of you not familiar, this involves a two step PCR protocol. The first round of PCR is carried out with your custom primers that have 5' overhangs complementary to the nextera indexes. The second round of PCR adds the indexes and adapters. The issue I am having is that my primer set contains inosine and so I can only get amplification using a Taq polymerase (Accuprime Taq in my case). This leaves my 1st PCR product with 3' A overhangs and I am not sure how this will effect the 2nd PCR step.
If I use a proofreading enzyme in the 2nd step, will this remove the 3' A's or do I need to include a specific step in my protocol to blunt my 1st round amplicons?
Could I accomplish blunting by using a proofreading polymerase and including a 72 degree incubation before the initial denaturing step in my 2nd round of PCR? Do I need to do a specific blunting step and then an additional PCR cleanup before round 2 of PCR?
Thanks!
Hi Julian Trachsel
If you want to prepare your pcr product of first round of amplification their there will be two option to do it
1. Use proof reading taq in both rounds of pcr amplification
2. U can amplified products of both the pcr amplification rounds by either end trimming or end filling by using normal taq (- proofreading)
Not sure it will affect anything since it is on the 3'end. I have not heard of problems of that sort. Just in case of amplicons it my be wise to use uridilated templates to prevent carryover - here is a kit that is supposed to do what you need:
KAPA HiFi HotStart Uracil+
ReadyMix PCR Kit
URACIL and UNG is what can help you in this situation. Kapa Mix has this combination for creating libraries.
Hi Julian,
I agree with Petre. The a overhangs should have no impact in the end. You can go about as planned. Once you do the second round of amplification, introducing the indexes, the new amplicons will not contain the 3' overhangs as as these are not included in the indexing primers.
Cheers,
Vegard
Hi Julian,
Did you manage to run your sequencing? If so, please can provide any advises, protocols or feedback?
Cheers,
Amine
Hey Amine,
Yes, the library prep and sequencing went well. As the people in this thread indicated the 3' overhang did not affect the 2nd step PCR. I was able to generate custom amplicon libraries and sequence them with minimal deviations from the recommended protocol (Nextera custom amplicon). This was several years ago at this point, so there may be better options available now
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