Authors
Abhishek D. Garg
Summary
‘Artificial/Chemical Gene-Synthesis’ has become an important contemporary technique, which has been refined several folds owing to application of Polymerase Chain Reaction (PCR) (Engels, 2005). This procedure has been used in past for artificially synthesizing a number of genes (Engels, 2005). The principle of Artificial Gene-Synthesis has been elucidated in Figure 1 (Casimiro et al, 1997; Engels, 2005; Engels & Uhlmann, 1989; Tsuchiya et al, 2006). Technically speaking, this procedure involves two phases i.e. upstream-phase and downstream-phase. In the upstream phase, the oligonucleotides (whose sequence is derived from target gene to be synthesized) are designed manually or in silico while in the downstream phase, these designed oligonucleotides are chemically synthesized and assembled together to give stable ds-DNA duplexes/target gene. The peculiarity of these oligonucleotides is that, each forward (5’→3’) oligonucleotide overlaps with the corresponding reverse (3’→5’) oligonucleotide in around 20 terminal nucleotide-bases, thus allowing formation of an annealed-product. Current protocol focuses on the upstream-phase since it’s a success-defining phase for artificial gene-synthesis. In the current protocol, various state-of-art bioinformatics tools have been brought together to provide a comprehensive-approach towards in silico designing of oligonucleotides. Further, for the first time, DNA/mRNA secondary-structure formation analysis has been included in such a protocol.Further details
The protocol was published on Protocol Exchange on 22 January 2009. To see the entire protocol, click on the source link.Advertisement
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