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Artificial Gene Synthesis

PostPosted: Sat Feb 25, 2012 6:17 pm
by Prasanth
Artificial gene synthesis is the process of synthesizing a gene in vitro without the need for initial template DNA samples. The main method is currently byoligonucleotide synthesis (also used for other applications) from digital genetic sequences and subsequent annealing of the resultant fragments. In contrast, naturalDNA replication requires existing DNA templates for synthesizing new DNA.

Synthesis of the first complete gene, a yeast tRNA, was demonstrated by Har Gobind Khorana and coworkers in 1972. Synthesis of the first peptide- and protein-coding genes was performed in the laboratories of Herbert Boyer and Alexander Markham, respectively.

Commercial gene synthesis services are now available from numerous companies worldwide, some of which have built their business model around this task.Current gene synthesis approaches are most often based on a combination of organic chemistry and molecular biological techniques and entire genes may be synthesized "de novo", without the need for precursor template DNA. Gene synthesis has become an important tool in many fields of recombinant DNA technology including heterologous gene expression, vaccine development, gene therapy and molecular engineering. The synthesis of nucleic acid sequences is often more economical than classical cloning and mutagenesis procedures. The market for gene synthesis was growing constantly over the past years. Experts estimated its volume to 40 Mio US-$ by the end of 2007.

Chemical synthesis of oligonucleotides

Oligonucleotides are chemically synthesized using nucleotides, called phosphoramidites, normal nucleotides which have protection groups: preventing amine, hydroxyl groups and phosphate groups interacting incorrectly. One phosphoramidite is added at a time, the product's 5' phosphate is deprotected and a new base is added and so on (backwards), at the end, all the protection groups are removed. Nevertheless, being a chemical process, several incorrect interactions occur leading to some defective products. The longer the oligonucleotide sequence that is being synthesized, the more defects there are, thus this process is only practical for producing short sequences of nucleotides. The current practical limit is about 200 bp for an oligonucleotide with sufficient quality to be used directly for a biological application. HPLC can be used to isolate products with the proper sequence. Meanwhile a large number of oligos can be synthesized in parallel on gene chips. For optimal performance in subsequent gene synthesis procedures they should be prepared individually and in larger scales.

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