Why is the ME-rev oligo on Tn5 phosphorylated?

[Adem80]

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]

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]

Thanks nucacidhunter.

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

[nucacidhunter]

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]

Thank you very much.

[Adem80]

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]

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

[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?

[luc]

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]

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

[Adem80]

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]

Ez-Tn5

Does anybody know the loading protocol for this enzyme?

[Adem80]

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.