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Are different assembly techniques possible?

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.

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