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  • 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.

  • #2
    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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    • #3
      Thanks nucacidhunter.

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

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      • #4
        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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        • #5
          Thank you very much.

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          • #6
            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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            • #7
              Any non-hot start polymerase will do the gap filling.

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              • #8
                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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                • #9
                  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, 05:01 PM.

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                  • #10
                    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

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                    • #11
                      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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                      • #12
                        Ez-Tn5

                        Does anybody know the loading protocol for this enzyme?

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                        • #13
                          Originally posted by SCIL2019 View Post
                          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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