Basically, there are three different approaches
for the assembly of synthetic genes. In the approach developed by
Khorana (Gupta et al., 1968) a series of sequentially overlapping
oligonucleotides are synthesised. After the annealing of the
oligos, a double stranded DNA fragment containing nicks on both
strands is formed. The nicks are sealed in a reaction with DNA
ligase, an enzyme that catalysis the formation of phosphodiester
bonds between the 5´-phosphate of one double strand oligonucleotide
fragment and the 3´-hydroxyl terminus on another adjacent double
strand oligonucleotide fragment.
An alternative strategy has been developed,
making use of the possibility of synthesising very long
oligonucleotide chains. In this approach (Rossi et al., 1982) two
oligonucleotides are constructed which upon annealing their 3´-ends
overlap. This construct is completed to a full length double strand
by a subsequent filling-in reaction using a DNA polymerase. After
polymerisation overhanging ends are generated on the double strand
fragment by digestion with an appropriate restriction enzyme.
Usually, all methods are followed by the
molecular cloning of the gene. Therefore it is necessary to
consider the cloning steps while developing the assembly strategy.
Larger genes can be divided in sub fragments that are assembled
separately. After sequence verification of the cloned sub
fragments, they are assembled to the full length construct.