Topoisomerase I mediated TA cloning: Difference between revisions
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==Topoisomerase site== | ==Topoisomerase site== | ||
===Topoisomerase=== | |||
Topoisomerase recognition site | |||
--------- | |||
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 | |||
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 26: | Line 72: | ||
**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 40: | 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> | ||
<biblio> | |||
#Heyman-GenomeRes-1999 pmid=10207160 | |||
</biblio> | |||
*Vectors pcDNA3.1/GS and pYES2/GS | *Vectors pcDNA3.1/GS and pYES2/GS | ||
Line 65: | Line 116: | ||
*Ligate on oligos (TOPO-H and TOPO-4) | *Ligate on oligos (TOPO-H and TOPO-4) | ||
**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 122: | ||
TOPO-H oligo | TOPO-H oligo | ||
----------------------------------------------- | ----------------------------------------------- | ||
5' N N N N N A | 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 | 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 130: | ||
TOPO-4 oligo HindIII overhang | TOPO-4 oligo HindIII overhang | ||
------------ ------- | ------------ ------- | ||
A G G G C G | 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 | 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 143: | ||
TOPO-H oligo | TOPO-H oligo | ||
------------------------------------------------- | ------------------------------------------------- | ||
5' N N N N N A | 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 | 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 151: | ||
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 | 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 | 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 162: | ||
==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? | |||
==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]] |
Latest revision as of 07:53, 25 July 2007
See also TOPO TA cloning.
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: 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
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
- 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
- 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).
- 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?
Notes
This approach to topoisomerase I mediated cloning has NOT been tested. This project is still a work in progress.
Additional references
Topoisomerase I
- Shuman S and Moss B. Identification of a vaccinia virus gene encoding a type I DNA topoisomerase. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7478-82. DOI:10.1073/pnas.84.21.7478 |
- Shuman S and Prescott J. Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. J Biol Chem. 1990 Oct 15;265(29):17826-36.
- Shuman S. DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992 Apr 25;267(12):8620-7.
- Shuman S. Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10104-8. DOI:10.1073/pnas.88.22.10104 |
- Shuman S. Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992 Aug 25;267(24):16755-8.
- Shuman S. DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J Biol Chem. 1992 Apr 25;267(12):8620-7.
- Shuman S. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem. 1994 Dec 23;269(51):32678-84.