Topo cloning

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See also TOPO TA cloning.

Topoisomerase site

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Topoisomerase

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   Topoisomerase recognition site
 ---------
    Topoisomerase nick site
          |
          V
5' C C C T T N N N N N N
3' G G G A A N N N N N N
        ^
        |
 Restriction enzyme must nick here

Offset nicking enzyme

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   Nt.BstNB I recognition site 
 ---------
           Nt.BstNB I nick site
                  |
                  V
5' G A G T C N N N N N 3'
3' C T C A G N N N N N 5'

Site for topoisomerase-mediated cloning

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Upstream of the BioBricks prefix

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   Topoisomerase recognition site
 ---------
          |
          V
5' C C C T T N N N G A C T C 3'
3' G G G A A N N N C T G A G 5'
        ^
        |
                 ---------
                 Nt.BstNB I recognition site

Downstream of BioBricks suffix

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   Nt.BstNB I recognition site
 ---------
                  |
                  V
5' G A G T C N N N A A G G G 3'
3' C T C A G N N N T T C C C 5'
                ^
                |
                 ---------
                 Topoisomerase recognition site

Possible enzymes to generate 3' T overhang

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None of these enzymes will work because it appears as if the topoisomerase enzyme needs some duplex DNA in order to function.

Each can accommodate the topoisomerase site CCCTT

  • BmrI
    • offset cutter that leaves 3' single base overhang
    • two sites in sopC (incD) repeat region of BBa_I50000
    • no sites in BBa_I50020
    • no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
    • pretty high activity in all 4 NEB buffers
  • BciVI
    • offset cutter that leaves 3' single base overhang
    • no sites in BBa_I50000
    • one site in BBa_I50020
    • no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
    • Austin says this is a very bad enzyme.
  • XcmI
    • long recognition sequence with internal N9 that leaves 3' single base overhang
    • no sites in BBa_I50000
    • no sites in BBa_I50020
    • no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
    • 100% activity only in NEBBuffer 2

Topoisomerase sites internal to vector components are not important because topoisomerase only operates near double stranded breaks.

Based on the preexistence of sites in the designed parts, XcmI looks like a good bet but it is not as robust to buffer conditions as BmrI.

Topoisomerase I

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Most of the papers reference Topoisomerase I from vaccinia. It seems to be available commercially at Epicentre. html protocol

  • Anselm Levskaya - I've gotten Vaccinia WR strain isolate and am presently cloning the topoisomerase IB gene out. Classically this is purified by using a phosphocellulose column but I'm going to just try a His-Tagged approach since it's a small protein.

Vector preparation

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Reference
<biblio>

  1. Heyman-GenomeRes-1999 pmid=10207160

</biblio>

  • Vectors pcDNA3.1/GS and pYES2/GS
               v
            HindIII site
            -----------
5' N N N N N  A A G C T T  N N N N N 3'
3' N N N N N  T T C G A A  N N N N N 5'
            -----------
            HindIII site
                     ^
  • Cut with HindIII
5' C C C T T  A 
3' G G G A A  T T C G A

           A G C T T  A A G G G 3'
                   A  T T C C C 5'
  • Ligate on oligos (TOPO-H and TOPO-4)
    • TOPO-H destroys the HindIII site
    • TOPO-4 provides recessed end
    • In the drawings below, " | " refers to a break in the backbone on that strand.
                  TOPO-H oligo
                -----------------------------------------------
5' N N N N N  A   A G C T   C G C C C T T A T T C C G A T A G T G
3' N N N N N  T   T C G A   G C G G G A
                -------   -----------
       HindIII overhang   TOPO-4 oligo


                      TOPO-4 oligo   HindIII overhang
                      ------------   -------
                      A G G G C  G   A G C T T   N N N N N 3'
G T G A T A C C T T A T T C C C G  C   T C G A A   N N N N N 5'
------------------------------------------------
TOPO-H oligo
  • Purify and cut again with HindIII to remove circular vector.
  • Add TOPO-5 oligo and topoisomerase.
    • Vaccinia topoisomerase I cleaves after and remains covalently attached to second T in CCCTT sequence.
                                           Topoisomerase nick
                                         v
                TOPO-H oligo
                -------------------------------------------------
5' N N N N N  A   A G C T   C G C C C T   T A T T C C G A T A G T G        3'
3' N N N N N  T   T C G A   G C G G G A | A T A A G G C T A T C A C A A C  5'
                -------   -----------   -------------------------------
       HindIII overhang   TOPO-4 oligo  TOPO-5 oligo


TOPO-5 oligo                      TOPO-4 oligo   HindIII overhang
-------------------------------   ------------   -------
C A A C A C T A T C G G A A T A | A G G G C  G   A G C T   T  N N N N N  3'
      G T G A T A C C T T A T   T C C C G  C   T C G A   A  N N N N N  5'
      ------------------------------------------------
      TOPO-H oligo
                           ^
                           Topoisomerase nick
  • Add TOPO-10X stop buffer
  • Purify away free oligonucleotides and unbound topoisomerase I

Questions

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  1. The authors add a primer TOPO-5 into the mix when they add the isomerase. It seems like this primer should not be necessary. Does the topoisomerase I "need" some duplex DNA extending out from where it will nick in order to cleave the backbone? Austin thinks most enzymes do need duplex DNA.
  2. How much of a performance hit will we take if we don't do all the purification steps they do ... it kind of seems like a lot of work (even though you only have to prep the vector once for multiple reactions).
  3. Can a restriction enzyme and topoisomerase I cleave simultaneously or will they occlude one another?
  4. Does TOPO TA cloning have a better success rate than TA cloning or is it just simpler?

Notes

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This approach to topoisomerase I mediated cloning has NOT been tested. This project is still a work in progress.

Additional references

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Topoisomerase I

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<biblio>

  1. Shuman-PNAS-1987 pmid=2823264
  2. Shuman-JBC-1990 pmid=2170398
  3. Shuman-JBC-1991 pmid=1314832
  4. Shuman-PNAS-1991 pmid=1658796
  5. Shuman-JBC-1992 pmid=1324909
  6. Shuman-JBC-1992b pmid=1314832
  7. Shuman-JBC-1994 pmid=7798275

</biblio>

Purpose

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Efficient cloning of PCR products.

Materials

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Procedure

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Reshma's procedure

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(Note that the kit details a slightly different procedure which may give superior efficiencies.)

  1. Mix 1 μL PCR product (amplified with Platinum PCR supermix) with 0.5 or 1μL TOPO vector (0.5μL should be sufficient.)
  2. Incubate 5 mins on the bench top
  3. Place on ice
  4. Transform

Notes

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  • Tom Knight claims you can cut the reaction volume in half and it will still work fine.
  • Use fresh PCR product for best results. (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
  • Your primers should NOT have 5' phosphates. They interfere with topoisomerase I.
  • From Tom Knight: As this article discusses, the 5' end of the PCR primer has a strong effect on the likelihood of 3' A addition to the PCR product when using Taq or Taq mixtures as enzymes during PCR. Since it is often necessary to add such an overhang to PCR products for cutting of an added cloning site, and since for such applications the sequence is immaterial, we should probably standardize that sequence to the A tail favoring sequence, GTTTCT for all PCR primers we make. This will favor a 3' A overhang on the PCR products, and allow TA cloning, or TOPO TA cloning of these products. Blunt end cloning could still be done by using Pfu or Phusion enzymes. I can't make any sense from the result about extending the primer sequence another base which is not specified. It seems to me this can not have any effect.
    • The 3' A overhang efficiency can be promoted even further by increasing the sequence length to GTTTCTT.
  • Reshma Shetty was generating some PCR fragments via annealing and primer extension and trying to cut with BioBricks enzymes and clone but the reactions were failing. Switching to TOPO TA cloning solved the problem.
  • In order for your part to be in BioBricks format after TOPO TA cloning, you must have the full BioBricks ends on your primers.
  • Austin Che observed anecdotally that he had a greater percentage of correct clones when he screened colonies from Kan plates rather than Amp plates.
  • Also see Knight:Addition_of_3'_A-overhangs.

Resources

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References

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<biblio>

  1. Brownstein-Biotechniques-1996 pmid=8780871
  2. Shuman-PNAS-1991 pmid=1658796
  3. Cheng-NAR-2000 pmid=10756188
  4. Shuman-JBC-1994 pmid=7798275
  5. Heyman-GenomeResearch-1999 pmid=10207160

</biblio>

Patents

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  1. US Patent 6,916,632; Methods and reagents for molecular cloning. (On topoisomerase-mediated cloning using a 5' overhang and blunt end.) pdf html
  2. US Patent 5,766,891; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase pdf html
  3. US Patent 6,653,106; Topoisomerase based ligation and cloning methods pdf
  4. US Patent 6,548,277; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase pdf html
  5. US Patent 5,487,993; Direct cloning of PCR amplified nucleic acids pdf html
  6. US Patent 5,827,657; Direct cloning of PCR amplified nucleic acids pdf html
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