Topoisomerase I mediated TA cloning

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Line 26: Line 26:
**one site in BBa_I50020
**one site in BBa_I50020
**no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
**no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
 +
**Austin says this is a very bad enzyme.
*[http://www.neb.com/nebecomm/products/productR0533.asp XcmI]
*[http://www.neb.com/nebecomm/products/productR0533.asp XcmI]
**long recognition sequence with internal N<sub>9</sub> that leaves 3' single base overhang
**long recognition sequence with internal N<sub>9</sub> that leaves 3' single base overhang
Line 65: Line 66:
*Ligate on oligos (TOPO-H and TOPO-4)
*Ligate on oligos (TOPO-H and TOPO-4)
-
**By ligate, I think the paper really means anneal ... i.e. the backbone bond is not restored.
 
**TOPO-H destroys the HindIII site
**TOPO-H destroys the HindIII site
**TOPO-4 provides recessed end
**TOPO-4 provides recessed end
Line 72: Line 72:
                   TOPO-H oligo
                   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
+
  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
+
  3' N N N N N  T  T C G A   G C G G G A
                   -------  -----------
                   -------  -----------
           HindIII overhang  TOPO-4 oligo
           HindIII overhang  TOPO-4 oligo
Line 80: Line 80:
                         TOPO-4 oligo  HindIII overhang
                         TOPO-4 oligo  HindIII overhang
                         ------------  -------
                         ------------  -------
-
                         A G G G C  G | A G C T T  N N N N N 3'
+
                         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'
+
  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
  TOPO-H oligo
Line 93: Line 93:
                   TOPO-H oligo
                   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'
+
  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'
+
  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
           HindIII overhang  TOPO-4 oligo  TOPO-5 oligo
Line 101: Line 101:
  TOPO-5 oligo                      TOPO-4 oligo  HindIII overhang
  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'
+
  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'
+
         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
         TOPO-H oligo
Line 112: Line 112:
==Questions==
==Questions==
-
#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?
+
#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.
#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.
#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.
#Can a restriction enzyme and topoisomerase I cleave simultaneously or will they occlude one another?
#Can a restriction enzyme and topoisomerase I cleave simultaneously or will they occlude one another?
 +
#Does TOPO TA cloning have a better success rate than TA cloning or is it just simpler?

Revision as of 12:38, 11 January 2006

See also TOPO TA cloning.

Contents

Topoisomerase site

Topoisomerase recognition site
---------
   Topoisomerase nick site
         |
         V
C C C T T N N N N N N
G G G A A N N N N N N
       ^
       |
Restriction enzyme must nick here

Possible enzymes to generate 3' T overhang

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

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

Vector preparation

Reference
J. A. Heyman, J. Cornthwaite, L. Foncerrada, J. R. Gilmore, E. Gontang, K. J. Hartman, C. L. Hernandez, R. Hood, H. M. Hull, W. Y. Lee, R. Marcil, E. J. Marsh, K. M. Mudd, M. J. Patino, T. J. Purcell, J. J. Rowland, M. L. Sindici, and J. P. Hoeffler. Genome-scale cloning and expression of individual open reading frames using topoisomerase i-mediated ligation. Genome Res, 9(1088-9051 (Print)):383–92, 1999. pubmed

  • 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

  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?
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