Just a technical question. During vector preparation in DNA extraction, we use dephosphorylase to prevent random ligation. However, after adding the vector and DNA sample, we use ligase to ligate the 2 strands together. As far as I know, ligase needs 3'-OH and 5'-PO4 for the ligation. However, the 5-PO4 is already removed due to the previous dephosphorylation. So how can the ligation still work?
It works because the molecules are double stranded. So if the vector has been dephosphorylated but the insert still has its terminal phosphates, then after ligation your will have one o the two strands ligated (at each end of the insert). So you get a ligated molecule with a nick at each junction that is repaired after transformation.
-------------------P-O--------------------------------- * -----------------------
-------------------- * ----------------------------------P-O---------------------
If both vector and insert were dephosphorylated then you would not get any ligation.
There are all sorts of DNA repair mechanisms in the cell that will fix It. It might get phosphorylated and lighted in the cell or more likely a gap repair mechanism will nibble back a few bases and resynthesize and seal.
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