A phospodiester bond is a covalent bond in which a phosphate group joins adjacent carbons through ester linkages. The bond is the result of a condensation reaction between a hydroxyl group of two sugar groups and a phosphate group. The diester bond between phosphoric acid and two sugar molecules in the DNA and RNA backbone links two nucelotides together to form oligonucleotide polymers. The phosphodiester bond links a 3′ carbon to a 5′ carbon in DNA and RNA.
(base)1-(sugar)-OH + HO-P(O)2-O-(sugar)-(base)2 
(base)1-(sugar)-O-P(O)2-O-(sugar)-(base)2
During the reaction of two of the hydroxyl groups in phosphoric acid with a hydroxyl group in two other molecules two ester bonds in a phosphodiester group are formed. A condensation reaction in which a water molecule is lost generates each ester bond. During polymerization of nucleotides to form nucleic acids, the hydroxyl group on the phosphate group attaches to the 3’ carbon of a sugar of one nucleotide to form an ester bond to the phosphate of another nucleotide. The reaction forms a phosphodiester linkage and eliminates a water molecule.
Figure 1: Phosphodiester bond formation in cells.
DNA and RNA polymerization occurs via the condensation of two monomers or a DNA or RNA strand and the condensation with an incoming nucleotide triphosphate. This condensation reaction is similar to peptide condensation reactions. The result is a single nucleic acid strand which is a phosphate-pentose polymer (this is a polyester) with purine and pyrimidine bases as side groups. The links between the nucleotides are called phosphodiester bonds. Regarding the chemical orientation, the 3’-end has a free hydroxyl group at the 3’-carbon of a sugar, and the 5’end has a free hydroxyl group or phosphate group at the 5’-carbon of a sugar. Since the synthesis proceeds from the 5’ to the 3’-end, according to convention sequences are written from in the 5’ -> 3′ direction. For example, AUG is assumed to be (5’)AUG(3’).
DNA polymerases catalyze the formation of polynucleotide chains through the addition of new nucleotides from incoming deoxynucleoside triphosphates. The polymerase reaction needs an appropriate DNA template to take place. Each incoming nucleoside triphosphate first forms a base pair with a base in the template. Next, the DNA polymerase links the incoming base with the predecessor in the chain. Therefore, DNA polymerases are template-directed enzymes.
When adding nucleotides to the 3′ end of a polynucleotide chain the polymerase catalyzes the nucleophilic attack of the 3′-hydroxyl group terminus of the polynucleotide chain on the α-phosphate group of the nucleoside triphosphate that is added. For the initiation of this reaction, DNA polymerases require a primer with a free 3′-hydroxyl group already base-paired to the template and cannot start from scratch by adding nucleotides to a free single-stranded DNA template. However, RNA polymerases can initiate RNA synthesis without a primer.
In gene cloning, a key step is to recombine the selected gene into a plasmid vector. The use of two different endonucleases that cleave on either side of the gene generating distinctive single-strand ends allows the isolation of gene on a restriction fragment. Directional cloning using two different enzymes allow the production of restriction fragments that have different noncomplementary overhangs at each end. The sticky ends of the fragments are rejoined with the complementary end in an opened up plasmid vector. The single-stranded overhang of a sticky end can form hydrogen bonds with the complementary nucleotides in the overhang of another fragment. A DNA ligase re-forms phosphodiester bonds between adjacent nucleotides. The ligation reaction links the deoxyribose-phosphate rails of the fragments into a stable double helix. This ligation reaction is another example of a condensation reaction.
