Why is the ME-rev oligo on Tn5 phosphorylated?

Adem80 · 02-20-2019, 10:42 AM

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.

nucacidhunter · 02-20-2019, 09:45 PM

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.

Adem80 · 02-20-2019, 10:25 PM

Thanks nucacidhunter.

But the Tm of this short fragment is 45°C. At 72°C it is probably detached from the transferred strand.

nucacidhunter · 02-21-2019, 02:18 AM

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.

Adem80 · 02-21-2019, 04:10 AM

Thank you very much.

Adem80 · 05-05-2019, 11:02 AM

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?

nucacidhunter · 05-05-2019, 04:17 PM

Any non-hot start polymerase will do the gap filling.

Adem80 · 05-05-2019, 10:35 PM

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?

luc · 05-06-2019, 05:58 PM

Quote:

Originally Posted by Adem80

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?)

SNPsaurus · 05-06-2019, 07:58 PM

This paper has some info on phosphate's role in transposition with an indirect role: https://www.ncbi.nlm.nih.gov/pubmed/18790806

Adem80 · 05-08-2019, 08:09 AM

Quote:

Originally Posted by SNPsaurus

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!

SCIL2019 · 10-01-2019, 10:56 AM

Does anybody know the loading protocol for this enzyme?

Adem80 · 04-16-2020, 05:03 AM

Quote:

Originally Posted by SCIL2019

Does anybody know the loading protocol for this enzyme?

Hi,

You can find it in the following publications:

Picelli et al., Tn5 transposase and tagmentation procedures for massively scaled sequencing projects, Genome Research, Vol. 24,
2033-2040, 2014.

Clark et al., Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-
seq), Nature Protocols, Vol. 12, 534-547, 2017.

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02-20-2019, 10:42 AM	#1
Adem80 Junior Member Location: Bonn Join Date: Aug 2018 Posts: 7	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.

05-06-2019, 07:58 PM	#10
SNPsaurus Registered Vendor Location: Eugene, OR Join Date: May 2013 Posts: 521	This paper has some info on phosphate's role in transposition with an indirect role: https://www.ncbi.nlm.nih.gov/pubmed/18790806 __________________ Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

10-01-2019, 10:56 AM	#12
SCIL2019 Junior Member Location: Melbourne, Australia Join Date: May 2019 Posts: 7	Ez-Tn5 Does anybody know the loading protocol for this enzyme?

02-20-2019, 09:45 PM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	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.

02-20-2019, 10:25 PM	#3
Adem80 Junior Member Location: Bonn Join Date: Aug 2018 Posts: 7	Thanks nucacidhunter. But the Tm of this short fragment is 45°C. At 72°C it is probably detached from the transferred strand.

02-21-2019, 02:18 AM	#4
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	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.

02-21-2019, 04:10 AM	#5
Adem80 Junior Member Location: Bonn Join Date: Aug 2018 Posts: 7	Thank you very much.

05-05-2019, 11:02 AM	#6
Adem80 Junior Member Location: Bonn Join Date: Aug 2018 Posts: 7	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?

05-05-2019, 04:17 PM	#7
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Any non-hot start polymerase will do the gap filling.

05-05-2019, 10:35 PM	#8
Adem80 Junior Member Location: Bonn Join Date: Aug 2018 Posts: 7	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?