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Old 06-18-2015, 11:11 AM   #1
Ingeneious
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Default End Repair + A-Tailing - how do they do it?

Some of the popular library prep kits out there (i.e. NEBNext Ultra and KAPA Hyper) are able to combine end repair and A-tailing into one step. I would like to do the same with minimal cost, but am not sure how they are doing it.

Both NEB and KAPA have the same thermocycler protocol:
  • 20°C for 30 min (Activation?)
  • 65°C for 30 min (Denaturation?)

End-repair can be done with some or a combination of the following enzymes:
  • DNA Polymerase I, Large (Klenow) Fragment (Active at 25°C for 15 min)
  • T4 DNA Polymerase (Active at 15°C for 12 min)
  • T4 Polynucleotide Kinase (Active at 37°C for 30 min)

A-Tailing can be done with some or a combination of the following enzymes:
  • Taq Polymerase (Active at 72°C for 20 min)
  • Klenow Fragment (3'→5' exo-) (Active at 37°C for 30 min)

Anyone know how they do it?
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Old 06-18-2015, 04:24 PM   #2
nucacidhunter
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I think they have Taq polymerase in End repair mix. During incubation at 65C Taq actively adds A to 3' ends, while the other mesophilic enzymes used for end repair and 5' phosphorylation are denatured and deactivated.
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Old 06-18-2015, 09:29 PM   #3
Ingeneious
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That is what I am thinking, too, but I have never seen a library prep protocol using it. Everyone seems to be using Klenow 3 - 5 exo for A-tailing and I have seen various combinations of enzymes for end repair.

Could Klenow be working simultaneously with the other enzymes and then they're all denatured?

Last edited by Ingeneious; 06-18-2015 at 09:32 PM.
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Old 06-18-2015, 09:49 PM   #4
luc
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At one point Lucigen claimed to have found a (propietary) enzyme (in hot springs) that is even better at A-tailing than Taq polymerase (in the presence of other nucelotides). This might have been licensed out. But in all likelihood it is simply Taq.
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Old 06-18-2015, 09:57 PM   #5
nucacidhunter
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That is less likely. For end repair (filling in 5’ overhang and removing 3’ overhang) two enzymatic activities are required so it is less likely to add 3’ A while another enzyme is removing 3’ overhangs. This can be tested by omitting incubation at 65C and immediately cleaning up end repair reaction followed by adapter ligation. Lack of 3’ A should significantly reduce ligation efficiency.
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Old 05-09-2016, 08:28 AM   #6
rcook34
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Hey all,

Sorry to reopen this older thread, but I was curious if anyone had anything else to add? I am very interested in this area, since my experiments using both NEB's and an in-house Klenow exo- enzyme for [email protected] degrees have given mostly negative results in terms of library yield.

Thanks!
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Old 07-28-2016, 01:48 PM   #7
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IME, Klenow Exo- is unnecessary and only was ever included because the very very first library kits from Solexa had it in there. T4 pol is a monster at end removal and fill in. The blunting phase of the reaction is over in <10 minutes (at least with clean DNA)

The best combination if you really want a one-pot reaction is T4 PNK + T4 Pol + Taq + dNTP. 20C is the ER phase. 65C is the heat-kill + Taq tailing phase. Non-templated addition is better at 65C than at 72C.

You WILL get lower A-tailing efficiency with Taq in the presence of all 4 nucleotides, but it's not that bad and probably worth the time savings.
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Old 09-15-2016, 09:42 PM   #8
zhengz
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The original paper combining several steps into one:

http://nar.oxfordjournals.org/content/38/13/e137.full
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Old 05-24-2018, 11:19 AM   #9
donnyKerabatsos
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This paper describes how to combine these steps in one reaction using individually-ordered (i.e. cheap) enzymes and buffers: T4 PNK, T4 polymerase and taq, same as @ECO suggests.

Library Preparation and Multiplex Capture for Massive Parallel Sequencing Applications Made Efficient and Easy
Mårten Neiman, Simon Sundling, Henrik Grönberg, Per Hall, Kamila Czene, Johan Lindberg , Daniel Klevebring
PLoS One
http://journals.plos.org/plosone/art...l.pone.0048616

Last edited by donnyKerabatsos; 05-24-2018 at 11:21 AM.
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