We will perform a simple cloning technique based on PCR. We have our covalently closed circular vector and an amplicon produced by asymmetric PCR that will serve as a megaprimer. Hence, it will result in a new vector with one nick in each strand, which after transformation will be closed in vivo. So the question is if in vivo ligation is stringently dependent on phosphorylated ends, and therefore a phosphorylation step is needed for the amplicons, or else we should request 5' end phosphorylated primers?
Based on this protocol: "Rapid Construction of Recombinant Plasmids
by QuickStep-Cloning"
Single- or double-stranded DNA with a 5'-hydroxyl terminus has to be phosphorylated prior to ligation as 5’ phosphate groups are required for ligation. Phosporylation of DNA ends with phosphate radioisotopes can also be used to label DNA in preparation of DNA for subsequent detection, isolation and sequencing applications.
Hi there, as the ligation reaction consists in the nucleophilic attack of the 3'OH on the P atom of the 5'phosphate group, 5' end has to be phosphorylated.
in vitro ligation would need the phosphorylated end but not the in vivo ligation.
@Avijit: The reaction mechanism of DNA ligase makes the presence of 5'phosphate compulsory as the first step in catalysis is activation of 5'phosphate by ATP. If 5'OH instead, no activation and no activation results in no ligation. For more info:
https://www.neb.com/applications/cloning-and-synthetic-biology/dna-ligation
Thank you Avijit, Dominque, and Murshidul, for your answers. I'm aware of the biochemical condition of the nucleic acid strands for the ligation reaction to occur. But I was wondering if E. coli's mechanism for strand break repair -besides ligation activity- readily included the phosphorylation step (and even physically linked to the same polypeptide as has been proposed for the RNA repair system). I searched for a polynucleotide kinase in E. coli genome databases, but I just found individual nucleotide kinases, which may suffice. At the end it is possible that it will be reflected in transformation efficiency.
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