Artificial gene synthesis

Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing.^[1] This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

Synthesis of the first complete gene, a yeast tRNA, was demonstrated by Har Gobind Khorana and coworkers in 1972.^[2] Synthesis of the first peptide- and protein-coding genes was performed in the laboratories of Herbert Boyer and Alexander Markham, respectively.^[3]^[4] More recently, artificial gene synthesis methods have been developed that will allow the assembly of entire chromosomes and genomes. The first synthetic yeast chromosome was synthesised in 2014, and entire functional bacterial chromosomes have also been synthesised.^[5] In addition, artificial gene synthesis could in the future make use of novel nucleobase pairs (unnatural base pairs).^[6]^[7]^[8]

^ Stein R (7 May 2015). "DNA 'Printing' A Big Boon To Research, But Some Raise Concerns". All Things Considered. National Public Radio.
^ Khorana HG, Agarwal KL, Büchi H, Caruthers MH, Gupta NK, Kleppe K, et al. (December 1972). "Studies on polynucleotides. 103. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast". Journal of Molecular Biology. 72 (2): 209–17. doi:10.1016/0022-2836(72)90146-5. PMID 4571075.
^ Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, Boyer HW (December 1977). "Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin". Science. 198 (4321): 1056–63. Bibcode:1977Sci...198.1056I. doi:10.1126/science.412251. PMID 412251.
^ Edge MD, Green AR, Heathcliffe GR, Meacock PA, Schuch W, Scanlon DB, et al. (August 1981). "Total synthesis of a human leukocyte interferon gene". Nature. 292 (5825): 756–62. Bibcode:1981Natur.292..756E. doi:10.1038/292756a0. PMID 6167861. S2CID 4330168.
^ Shukman D (2014-03-27). "Synthetic DNA advance is hailed". BBC News. Retrieved 2020-04-11.
^ Kimoto M, Yamashige R, Matsunaga K, Yokoyama S, Hirao I (May 2013). "Generation of high-affinity DNA aptamers using an expanded genetic alphabet". Nature Biotechnology. 31 (5): 453–7. doi:10.1038/nbt.2556. PMID 23563318. S2CID 23329867.
^ Malyshev DA, Dhami K, Quach HT, Lavergne T, Ordoukhanian P, Torkamani A, Romesberg FE (July 2012). "Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet". Proceedings of the National Academy of Sciences of the United States of America. 109 (30): 12005–10. Bibcode:2012PNAS..10912005M. doi:10.1073/pnas.1205176109. PMC 3409741. PMID 22773812.
^ Malyshev DA, Dhami K, Lavergne T, Chen T, Dai N, Foster JM, et al. (May 2014). "A semi-synthetic organism with an expanded genetic alphabet". Nature. 509 (7500): 385–8. Bibcode:2014Natur.509..385M. doi:10.1038/nature13314. PMC 4058825. PMID 24805238.

[1] Stein R (7 May 2015). "DNA 'Printing' A Big Boon To Research, But Some Raise Concerns". All Things Considered. National Public Radio.

[Khorana722-2] Khorana HG, Agarwal KL, Büchi H, Caruthers MH, Gupta NK, Kleppe K, et al. (December 1972). "Studies on polynucleotides. 103. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast". Journal of Molecular Biology. 72 (2): 209–17. doi:10.1016/0022-2836(72)90146-5. PMID 4571075.

[Itakura772-3] Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, Boyer HW (December 1977). "Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin". Science. 198 (4321): 1056–63. Bibcode:1977Sci...198.1056I. doi:10.1126/science.412251. PMID 412251.

[Edge812-4] Edge MD, Green AR, Heathcliffe GR, Meacock PA, Schuch W, Scanlon DB, et al. (August 1981). "Total synthesis of a human leukocyte interferon gene". Nature. 292 (5825): 756–62. Bibcode:1981Natur.292..756E. doi:10.1038/292756a0. PMID 6167861. S2CID 4330168.

[Shukman-5] Shukman D (2014-03-27). "Synthetic DNA advance is hailed". BBC News. Retrieved 2020-04-11.

[6] Kimoto M, Yamashige R, Matsunaga K, Yokoyama S, Hirao I (May 2013). "Generation of high-affinity DNA aptamers using an expanded genetic alphabet". Nature Biotechnology. 31 (5): 453–7. doi:10.1038/nbt.2556. PMID 23563318. S2CID 23329867.

[Malyshev_PNAS_201207242-7] Malyshev DA, Dhami K, Quach HT, Lavergne T, Ordoukhanian P, Torkamani A, Romesberg FE (July 2012). "Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet". Proceedings of the National Academy of Sciences of the United States of America. 109 (30): 12005–10. Bibcode:2012PNAS..10912005M. doi:10.1073/pnas.1205176109. PMC 3409741. PMID 22773812.

[NATJ-201405072-8] Malyshev DA, Dhami K, Lavergne T, Chen T, Dai N, Foster JM, et al. (May 2014). "A semi-synthetic organism with an expanded genetic alphabet". Nature. 509 (7500): 385–8. Bibcode:2014Natur.509..385M. doi:10.1038/nature13314. PMC 4058825. PMID 24805238.

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