Topoisomerase I mediated TA cloning

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Questions)
Current revision (09:53, 25 July 2007) (view source)
 
(9 intermediate revisions not shown.)
Line 2: Line 2:
==Topoisomerase site==
==Topoisomerase site==
-
Topoisomerase recognition site
+
 
-
---------
+
===Topoisomerase===
-
    Topoisomerase nick site
+
   
-
          |
+
    Topoisomerase recognition site
-
          V
+
    ---------
-
  C C C T T N N N N N N
+
      Topoisomerase nick site
-
  G G G A A N N N N N N
+
            |
-
        ^
+
            V
-
        |
+
  5' C C C T T N N N N N N
-
Restriction enzyme must nick here
+
  3' G G G A A N N N N N N
 +
          ^
 +
          |
 +
    Restriction enzyme must nick here
 +
 
 +
===Offset nicking enzyme===
 +
 
 +
*[http://www.neb.com/nebecomm/products/productR0607.asp Nt.BstNB I]: offset nicking enzyme from NEB.
 +
 
 +
    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===
 +
 
 +
====Upstream of the BioBricks prefix====
 +
 
 +
    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====
 +
 
 +
    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==
==Possible enzymes to generate 3' T overhang==
 +
 +
'''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
Each can accommodate the topoisomerase site CCCTT
*[http://www.neb.com/nebecomm/products/productR0600.asp BmrI]
*[http://www.neb.com/nebecomm/products/productR0600.asp BmrI]
Line 41: Line 87:
Most of the papers reference Topoisomerase I from vaccinia.  It seems to be available commercially at Epicentre. [http://www.epibio.com/item.asp?ID=274&CatID=112 html] [http://www.epibio.com/pdftechlit/108pl092.pdf protocol]
Most of the papers reference Topoisomerase I from vaccinia.  It seems to be available commercially at Epicentre. [http://www.epibio.com/item.asp?ID=274&CatID=112 html] [http://www.epibio.com/pdftechlit/108pl092.pdf 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==
==Vector preparation==
'''Reference'''<br>
'''Reference'''<br>
-
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. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10207160 pubmed]
+
<biblio>
 +
#Heyman-GenomeRes-1999 pmid=10207160
 +
</biblio>
*Vectors pcDNA3.1/GS and pYES2/GS
*Vectors pcDNA3.1/GS and pYES2/GS
Line 116: Line 166:
#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?
#Does TOPO TA cloning have a better success rate than TA cloning or is it just simpler?
 +
 +
==Notes==
 +
<font color=red>'''This approach to topoisomerase I mediated cloning has NOT been tested.  This project is still a work in progress.</font>
 +
 +
==Additional references==
 +
 +
===Topoisomerase I===
 +
<biblio>
 +
#Shuman-PNAS-1987 pmid=2823264
 +
#Shuman-JBC-1990 pmid=2170398
 +
#Shuman-JBC-1991 pmid=1314832
 +
#Shuman-PNAS-1991 pmid=1658796
 +
#Shuman-JBC-1992 pmid=1324909
 +
#Shuman-JBC-1992b pmid=1314832
 +
#Shuman-JBC-1994 pmid=7798275
 +
</biblio>
 +
 +
[[Category:In vitro]] [[Category:DNA]]

Current revision

See also TOPO TA cloning.

Contents

Topoisomerase site

Topoisomerase

   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

   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

Upstream of the BioBricks prefix

   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

   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

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

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

Reference

Error fetching PMID 10207160:
  1. Error fetching PMID 10207160: [Heyman-GenomeRes-1999]
  • 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?

Notes

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

Additional references

Topoisomerase I

Error fetching PMID 2823264:
Error fetching PMID 2170398:
Error fetching PMID 1314832:
Error fetching PMID 1658796:
Error fetching PMID 1324909:
Error fetching PMID 1314832:
Error fetching PMID 7798275:
  1. Error fetching PMID 2823264: [Shuman-PNAS-1987]
  2. Error fetching PMID 2170398: [Shuman-JBC-1990]
  3. Error fetching PMID 1314832: [Shuman-JBC-1991]
  4. Error fetching PMID 1658796: [Shuman-PNAS-1991]
  5. Error fetching PMID 1324909: [Shuman-JBC-1992]
  6. Error fetching PMID 1314832: [Shuman-JBC-1992b]
  7. Error fetching PMID 7798275: [Shuman-JBC-1994]
All Medline abstracts: PubMed HubMed
Personal tools