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Old 02-20-2019, 09:42 AM   #1
Adem80
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Default Why is the ME-rev oligo on Tn5 phosphorylated?

Hello all,

Does anyone know why the oligo named ME-rev, annealed to ME-A and ME-B before being loaded on the Tn5, is phosphorylated at the 5' end ([phos]CTGTCTCTTATACACATCT)?

Thanks a lot.
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Old 02-20-2019, 08:45 PM   #2
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Nextera (modified Tn5 transposase) inserts ME sequences by cut and paste mechanism and leaves a 9 base gap at the 3' end of insertion site. The gap is filled during 3 min incubation at 72C at PCR step. If there is no 5' phosphate there will be a nick and PCR will fail.
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Old 02-20-2019, 09:25 PM   #3
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Thanks nucacidhunter.

But the Tm of this short fragment is 45C. At 72C it is probably detached from the transferred strand.
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Old 02-21-2019, 01:18 AM   #4
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The calculated Tm is correct. Empirically it is difficult to dissociate that oligo. For an application I tried to inhibit gap filing by both heat and a chemical method. PCR yield was decreased by 65% but was not inhibited completely.
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Old 02-21-2019, 03:10 AM   #5
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Thank you very much.
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Old 05-05-2019, 10:02 AM   #6
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Hello,

One further question about this oligo. Which enzyme is doing the ligation at the phosphorylated base during the gap filling? As far as I know, the DNA polymerase does not have this property of ligation. We use KAPA HiFi mix or NEBNext HiFi mix for the PCR. Do these contain a DNA ligase?
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Old 05-05-2019, 03:17 PM   #7
nucacidhunter
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Any non-hot start polymerase will do the gap filling.
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Old 05-05-2019, 09:35 PM   #8
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But I mean, the 3' end of the nucleotide in the gap should be ligated to the phosphorylated 5' end of the oligo, I believe that's the reason why it should be phosphorylated, isn't it?
Can polymerases ligate?
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Old 05-06-2019, 04:58 PM   #9
luc
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Quote:
Originally Posted by Adem80 View Post
But I mean, the 3' end of the nucleotide in the gap should be ligated to the phosphorylated 5' end of the oligo, I believe that's the reason why it should be phosphorylated, isn't it?
Can polymerases ligate?
I am just speculating. AFAIU, the remaining oligos will be kicked off by nick-translation/strand-displacement in addition to the gap filling; I assume there is no ligation happening. (Perhaps the phosphate group is required for the transposase loading instead?)

Last edited by luc; 05-06-2019 at 05:01 PM.
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Old 05-06-2019, 06:58 PM   #10
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This paper has some info on phosphate's role in transposition with an indirect role: https://www.ncbi.nlm.nih.gov/pubmed/18790806
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Old 05-08-2019, 07:09 AM   #11
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Quote:
Originally Posted by SNPsaurus View Post
This paper has some info on phosphate's role in transposition with an indirect role: https://www.ncbi.nlm.nih.gov/pubmed/18790806
Very good explanation indeed. Thanks for sharing the paper!
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Old 10-01-2019, 09:56 AM   #12
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Default Ez-Tn5

Does anybody know the loading protocol for this enzyme?
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