Before you go wild with your imagination to what 'TOPO' is, its an acronym of DNA #Topoisomerase I! #TOPOTAcloning is the fastest #restrictionendonuclease and #ligase-free cloning method and is reliable too!
Principle
In continuity to the principle of #TAcloning, the #TaqPolymerase's non template-dependent #terminaltransferase activity adds a 3'-#Adenosine residue to the insert PCR product at both its ends.
The TOPO vector on the other hand has a single #Thymine overhang with Vaccinia virus DNA Topoisomerase I covalently attached to it. The Topoisomerase I here acts both as a #restrictionendonuclease as well as a #ligase. It linearizes the vector by introducing a single-strand break at the site 5´-(C/T)CCTT-3', conserving the energy in a covalent bond between the now free 3'-PO4 and Tyr-274 of #Topoisomerase I. On encountering the 5'-OH from the insert DNA, the earlier covalent bond breaks, relegating the DNA molecules and releasing the Topoisomerase I.
Proocol
FIGURE: The #TOPOTAcloning schematic protocol.
This is one of the simplest protocol involving barely 4 components, your insert, TOPO vector, salt solution (provided) and maybe water.
Make the mix and keep at room temperature for 5-10 mins.
Place the mix on ice if transforming or at -20C for future transformations.
Maybe throw a flying kiss to the incubator (!)
Merits
Its the fastest #restrictionendonuclease and #ligase-free cloning method, with a recommended incubation time of 5 mins although 30 seconds can also make you rush for your #compcells!
Its #restrictionendonuclease-free, so you don't need to look for cut sites in your insert DNA!
It is very #specific due to complementary mononucleotide overhangs!
PCR product insertion sites are flanked by #EcoRI sites in both vectors for easy excision of inserts and ligation to vector of choice.
Demerits
It is #expensive, time surely does come with money (preachy?!)
As #Invitrogen is the only supplier of this kit, there's no choice of vectors but the two supplied by them.
Applications
#Subcloning of your insert into the vector of choice is the most popular application. pCR2.1-TOPO vector has 15 cut sites to add convenience here.
#Sequencing of your insert can easily be done with the supplied primers viz. M13 forward and reverse. pCR4-TOPO vector has a total of 4 priming sites including T3 and T7 for proper identification of direction of cloning.
More to look into:
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