Topoisomerase I mediated TA cloning: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
Line 92: | Line 92: | ||
==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 |
Revision as of 10:20, 2 January 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?