Cloning 3 Flashcards
TA cloning
Taq polymerase has a terminal transferase activity that adds a single adenosine to the 3’ end of PCR amplified fragments
Describe TA cloning
Taq polymerase adds 3’ A overhangs on both ends of PCR amplified PCR products. A vector with 3’ T overhangs will anneal. T4 ligase
Caveat of TA cloning
Polymerases that have proofreading will not leave 3’ A overhangs. Thus PCR products will need to be blunt end ligated into a vector or A’s added with taq pol after regular PCR in a seperate extension rxn
TOPO TA cloning
Topoisomerase binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5’ CCCTT in one strand. The energy from the broken bond is conserved by the formation of a covalent bond between the 3’PO4 of the cleaved strand and tyr274 on the Topoisomerase 1. The phsopho tyrosyl bond can be attacked by the 5’PH of the original cleaved strand to reverse the rxn and release the TOPO1 enzzyme
Argue both ways that TOPO TA cloning is ligase free
It has ligase in that topoisomerase is used to attach the DNA so is re-ligating
It is ligase free because it does not require additional ligase
Gibson assembly
Uses three enzymes to join two or more sequences of DNA that must have 40bp at their joining point
Gibson assembly protocol. What 3 enxymes are required?
T5 exonuclease
Phusion polymerase
Taq ligase
What does T5 exonuclease so
chews back the 5’ ends of the DNA fragments
Phusion DNA polymerase
Fills in gaps and extends from the 3’ end and does not interfere with the T% exonuclease at the 5’ end
Taq ligase
Once the gaps are filled in by phusion polymerase taq ligase ligates DNA
Advantages of Gibson
You can assmeble several framgemnts at once
No extraneuos seq are introduced
Disadvantages of gibson
Seq of your fragments must bee known
Custom primers have to be designed
