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On Oct 6, 2007, [[Craig Venter]] announced in an interview with UK's ''[[The Guardian]]'' newspaper that the same team had synthesized a modified version of the single [[chromosome]] of ''[[Mycoplasma genitalium]]'' using chemicals. The chromosome was modified to eliminate all genes which tests in live bacteria had shown to be unnecessary. The next planned step in this ''minimal genome project'' is to transplant the synthesized minimal genome into a bacterial cell with its old DNA removed; the resulting bacterium will be called ''[[Mycoplasma laboratorium]]''. The next day the Canadian [[bioethics]] group, [[ETC Group]] issued a statement through their representative, [[Pat Roy Mooney|Pat Mooney]], saying Venter's "creation" was "a chassis on which you could build almost anything". The synthesized genome had not yet been transplanted into a working cell.<ref>{{cite news|url=http://www.guardian.co.uk/science/2007/oct/06/genetics.climatechange|publisher=The Guardian|date=2009-10-06|accessdate=2010-05-22|title=I am creating artificial life, declares US gene pioneer | location=London | first=Ed | last=Pilkington| archiveurl= http://web.archive.org/web/20100528172927/http://www.guardian.co.uk/science/2007/oct/06/genetics.climatechange| archivedate= 28 May 2010 <!--DASHBot-->| deadurl= no}}</ref>
On Oct 6, 2007, [[Craig Venter]] announced in an interview with UK's ''[[The Guardian]]'' newspaper that the same team had synthesized a modified version of the single [[chromosome]] of ''[[Mycoplasma genitalium]]'' using chemicals. The chromosome was modified to eliminate all genes which tests in live bacteria had shown to be unnecessary. The next planned step in this ''minimal genome project'' is to transplant the synthesized minimal genome into a bacterial cell with its old DNA removed; the resulting bacterium will be called ''[[Mycoplasma laboratorium]]''. The next day the Canadian [[bioethics]] group, [[ETC Group]] issued a statement through their representative, [[Pat Roy Mooney|Pat Mooney]], saying Venter's "creation" was "a chassis on which you could build almost anything". The synthesized genome had not yet been transplanted into a working cell.<ref>{{cite news|url=http://www.guardian.co.uk/science/2007/oct/06/genetics.climatechange|publisher=The Guardian|date=2009-10-06|accessdate=2010-05-22|title=I am creating artificial life, declares US gene pioneer | location=London | first=Ed | last=Pilkington| archiveurl= http://web.archive.org/web/20100528172927/http://www.guardian.co.uk/science/2007/oct/06/genetics.climatechange| archivedate= 28 May 2010 <!--DASHBot-->| deadurl= no}}</ref>


On May 21, 2010, [[Science (journal)|''Science'']] reported that the Venter group had successfully synthesized the genome of the bacterium ''Mycoplasma mycoides'' from a computer record, and transplanted the synthesized genome into the existing cell of a ''Mycoplasma capricolum'' bacterium that had had its DNA removed. The "synthetic" bacterium was viable, i.e. capable of replicating billions of times. The team had originally planned to use the ''M. genitalium'' bacterium they had previously been working with, but switched to ''M. mycoides'' because the latter bacterium grows much faster, which translated into quicker experiments.<ref>{{cite web |url=http://www.sciencemag.org/cgi/reprint/328/5981/958.pdf |title=Synthetic Genome Brings New Life to Bacterium |accessdate=2010-05-21 | work=Science | date=2010-05-21| archiveurl= http://web.archive.org/web/20100525181700/http://www.sciencemag.org/cgi/reprint/328/5981/958.pdf| archivedate= 25 May 2010 <!--DASHBot-->| deadurl= no}}</ref> Venter describes it as "the first species.... to have its parents be a computer".<ref>{{cite news |url=http://news.bbc.co.uk/1/hi/sci/tech/8695992.stm |title=How scientists made 'artificial life' |accessdate=2010-05-21 | work=BBC News | date=2010-05-20| archiveurl= http://web.archive.org/web/20100523015837/http://news.bbc.co.uk/1/hi/sci/tech/8695992.stm?| archivedate= 23 May 2010 <!--DASHBot-->| deadurl= no}}</ref> The transformed bacterium is dubbed "[[Synthia]]" by ETC. A Venter spokesperson has declined to confirm any breakthrough at the time of this writing.{{fact}}
On May 21, 2010, [[Science (journal)|''Science'']] reported that the Venter group had successfully synthesized the genome of the bacterium ''Mycoplasma mycoides'' from a computer record, and transplanted the synthesized genome into the existing cell of a ''Mycoplasma capricolum'' bacterium that had had its DNA removed. The "synthetic" bacterium was viable, i.e. capable of replicating billions of times. The team had originally planned to use the ''M. genitalium'' bacterium they had previously been working with, but switched to ''M. mycoides'' because the latter bacterium grows much faster, which translated into quicker experiments.<ref>{{cite web |url=http://www.sciencemag.org/cgi/reprint/328/5981/958.pdf |title=Synthetic Genome Brings New Life to Bacterium |accessdate=2010-05-21 | work=Science | date=2010-05-21| archiveurl= http://web.archive.org/web/20100525181700/http://www.sciencemag.org/cgi/reprint/328/5981/958.pdf| archivedate= 25 May 2010 <!--DASHBot-->| deadurl= no}}</ref> Venter describes it as "the first species.... to have its parents be a computer".<ref>{{cite news |url=http://news.bbc.co.uk/1/hi/sci/tech/8695992.stm |title=How scientists made 'artificial life' |accessdate=2010-05-21 | work=BBC News | date=2010-05-20| archiveurl= http://web.archive.org/web/20100523015837/http://news.bbc.co.uk/1/hi/sci/tech/8695992.stm?| archivedate= 23 May 2010 <!--DASHBot-->| deadurl= no}}</ref> The transformed bacterium is dubbed "[[Synthia]]" by ETC. A Venter spokesperson has declined to confirm any breakthrough at the time of this writing.{{fact|date=April 2013}}


==XNA==
==XNA==

Revision as of 10:42, 11 April 2013

Artificial gene synthesis is a method in synthetic biology that is used to create artificial genes in the laboratory. Based on solid-phase DNA synthesis, it differs from molecular cloning and polymerase chain reaction (PCR) in that the user does not have to begin with preexisting DNA sequences. Therefore, it is possible to make a completely synthetic double-stranded DNA molecule with no apparent limits on either nucleotide sequence or size. The method has been used to generate functional bacterial chromosomes containing approximately one million base pairs.

Synthesis of the first complete gene, a yeast tRNA, was demonstrated by Har Gobind Khorana and coworkers in 1972.[1] Synthesis of the first peptide- and protein-coding genes was performed in the laboratories of Herbert Boyer and Alexander Markham, respectively.[2][3]

Commercial gene synthesis services are now available from numerous companies worldwide, some of which have built their business model around this task.[4] 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.

Gene optimization

While the ability to make increasingly long stretches of DNA efficiently and at lower prices is a technological driver of this field, increasingly attention is being focused on improving the design of genes for specific purposes. Early in the genome sequencing era, gene synthesis was used as an (expensive) source of cDNA's that were predicted by genomic or partial cDNA information but were difficult to clone. As higher quality sources of sequence verified cloned cDNA have become available, this practice has become less urgent.

Producing large amounts of protein from gene sequences (or at least the protein coding regions of genes, the open reading frame) found in nature can sometimes prove difficult and is a problem of sufficient impact that scientific conferences have been devoted to the topic.All[5][6] Many of the most interesting proteins sought by molecular biologist are normally regulated to be expressed in very low amounts in wild type cells. Redesigning these genes offers a means to improve gene expression in many cases. Rewriting the open reading frame is possible because of the degeneracy of the genetic code. Thus it is possible to change up to about a third of the nucleotides in an open reading frame and still produce the same protein. The available number of alternate designs possible for a given protein is astronomical. For a typical protein sequence of 300 amino acids there are over 10150 codon combinations that will encode an identical protein. Using optimization methods such as replacing rarely used codons with more common codons sometimes have a dramatic effects. Further optimizations such as removing RNA secondary structures can also be included. At least in the case of E. coli, protein expression is maximized by predominantly using codons corresponding to tRNA's that retain amino acid charging during starvation.[7] Computer programs are written to perform these, and other simultaneous optimizations are used to handle the enormous complexity of the task.[8] A well optimized gene can improve protein expression 2 to 10 fold, and in some cases more than 100 fold improvements have been reported. Because of the large numbers of nucleotide changes made to the original DNA sequence, the only practical way to create the newly designed genes is to use gene synthesis.

Standard methods

Chemical synthesis of oligonucleotides

Main article: Oligonucleotide synthesis

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.

Annealing based connection of oligonucleotides

Usually, a set of individually designed oligonucleotides is made on automated solid-phase synthesizers, purified and then connected by specific annealing and standard ligation or polymerase reactions. To improve specificity of oligonucleotide annealing, the synthesis step relies on a set of thermostable DNA ligase and polymerase enzymes. To date, several methods for gene synthesis have been described, such as the ligation of phosphorylated overlapping oligonucleotides,[1][2] the Fok I method[3] and a modified form of ligase chain reaction for gene synthesis. Additionally, several PCR assembly approaches have been described.[9] They usually employ oligonucleotides of 40-50 nt long that overlap each other. These oligonucleotides are designed to cover most of the sequence of both strands, and the full-length molecule is generated progressively by overlap extension (OE) PCR,[9] thermodynamically balanced inside-out (TBIO) PCR[10] or combined approaches.[11] The most commonly synthesized genes range in size from 600 to 1,200 bp although much longer genes have been made by connecting previously assembled fragments of under 1,000 bp. In this size range it is necessary to test several candidate clones confirming the sequence of the cloned synthetic gene by automated sequencing methods.

Limitations

Moreover, because the assembly of the full-length gene product relies on the efficient and specific alignment of long single stranded oligonucleotides, critical parameters for synthesis success include extended sequence regions comprising secondary structures caused by inverted repeats, extraordinary high or low GC-content, or repetitive structures. Usually these segments of a particular gene can only be synthesized by splitting the procedure into several consecutive steps and a final assembly of shorter sub-sequences, which in turn leads to a significant increase in time and labor needed for its production. The result of a gene synthesis experiment depends strongly on the quality of the oligonucleotides used. For these annealing based gene synthesis protocols, the quality of the product is directly and exponentially dependent on the correctness of the employed oligonucleotides. Alternatively, after performing gene synthesis with oligos of lower quality, more effort must be made in downstream quality assurance during clone analysis, which is usually done by time-consuming standard cloning and sequencing procedures. Another problem associated with all current gene synthesis methods is the high frequency of sequence errors because of the usage of chemically synthesized oligonucleotides. The error frequency increases with longer oligonucleotides, and as a consequence the percentage of correct product decreases dramatically as more oligonucleotides are used. The mutation problem could be solved by shorter oligonucleotides used to assemble the gene. However, all annealing based assembly methods require the primers to be mixed together in one tube. In this case, shorter overlaps do not always allow precise and specific annealing of complementary primers, resulting in the inhibition of full length product formation. Manual design of oligonucleotides is a laborious procedure and does not guarantee the successful synthesis of the desired gene. For optimal performance of almost all annealing based methods, the melting temperatures of the overlapping regions are supposed to be similar for all oligonucleotides. The necessary primer optimization should be performed using specialized oligonucleotide design programs. Several solutions for automated primer design for gene synthesis have been presented so far.[12][13][14]

Error correction procedures

To overcome problems associated with oligonucleotide quality several elaborate strategies have been developed, employing either separately prepared fishing oligonucleotides,[15] mismatch binding enzymes of the mutS family[16] or specific endonucleases from bacteria or phages.[17] Nevertheless, all these strategies increase time and costs for gene synthesis based on the annealing of chemically synthesized oligonucleotides.

Massively parallel sequencing has also been used as a tool to screen complex oligonucleotide libraries and enable the retrieval of accurate molecules. In one approach, oligonucleotides are sequenced on the 454 pyrosequencing platform and a robotic system images and picks individual beads corresponding to accurate sequence.[18] In another approach, a complex oligonucleotide library is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by dial-out PCR.[19]

Increasingly, genes are ordered in sets including functionally related genes or multiple sequence variants on a single gene. Virtually all of the therapeutic proteins in development, such as monoclonal antibodies, are optimized by testing many gene variants for improved function or expression.

Applications

Major applications of synthetic genes include synthesis of DNA sequences identified by high throughput sequencing but never cloned into plasmids and the ability to safely obtain genes for vaccine research without the need to grow the full pathogens. Digital manipulation of digital genetic code before synthesis into DNA can be used to optimize protein expression in a particular host, or remove non-functional segments in order to facilitate further replication of the DNA.


Synthesis of DNA allows DNA digital data storage.

Entire genomes

Synthia and Mycoplasma laboratorium

Main article: Mycoplasma laboratorium

On June 28, 2007, a team at the J. Craig Venter Institute published an article in Science Express, saying that they had successfully transplanted the natural DNA from a Mycoplasma mycoides bacterium into a Mycoplasma capricolum cell, creating a bacterium which behaved like a M. mycoides.[20]

On Oct 6, 2007, Craig Venter announced in an interview with UK's The Guardian newspaper that the same team had synthesized a modified version of the single chromosome of Mycoplasma genitalium using chemicals. The chromosome was modified to eliminate all genes which tests in live bacteria had shown to be unnecessary. The next planned step in this minimal genome project is to transplant the synthesized minimal genome into a bacterial cell with its old DNA removed; the resulting bacterium will be called Mycoplasma laboratorium. The next day the Canadian bioethics group, ETC Group issued a statement through their representative, Pat Mooney, saying Venter's "creation" was "a chassis on which you could build almost anything". The synthesized genome had not yet been transplanted into a working cell.[21]

On May 21, 2010, Science reported that the Venter group had successfully synthesized the genome of the bacterium Mycoplasma mycoides from a computer record, and transplanted the synthesized genome into the existing cell of a Mycoplasma capricolum bacterium that had had its DNA removed. The "synthetic" bacterium was viable, i.e. capable of replicating billions of times. The team had originally planned to use the M. genitalium bacterium they had previously been working with, but switched to M. mycoides because the latter bacterium grows much faster, which translated into quicker experiments.[22] Venter describes it as "the first species.... to have its parents be a computer".[23] The transformed bacterium is dubbed "Synthia" by ETC. A Venter spokesperson has declined to confirm any breakthrough at the time of this writing.[citation needed]

XNA

An artificial genetic material called XNA (xeno-nucleic acid) was reported to be capable of replication in the test tube much like real DNA. In an article published in Science, researchers at the Laboratory of Molecular Biology showed that the synthetic XNA fragments can replicate and evolve with desired properties. XNAs maintain the same four-letter nucleobases but with modification to the double helix structure to add properties such as acid resistance. Scientists touted that ‘this brings us one big step closer to artificial life’, since the XNAs are showed to possess the properties of ‘heredity and evolution, two hallmarks of life’. At the more near termed future, XNAs could form a new platform for designer drugs and diagnostic tools to treat a variety of diseases.[24][25]

See also

Notes

  1. ^ a b Khorana HG; Agarwal KL; Büchi H; et al. (1972). "Studies on polynucleotides. 103. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast". J. Mol. Biol. 72 (2): 209–217. doi:10.1016/0022-2836(72)90146-5. PMID 4571075. {{cite journal}}: Unknown parameter |author-separator= ignored (help); Unknown parameter |month= ignored (help)
  2. ^ a b Itakura K; Hirose T; Crea R; et al. (1977). "Expression in Escherichia coli of a chemically synthesized gene for the hormone somatostatin". Science. 198 (4321): 1056–1063. Bibcode:1977Sci...198.1056I. doi:10.1126/science.412251. PMID 412251. {{cite journal}}: Unknown parameter |author-separator= ignored (help); Unknown parameter |month= ignored (help)
  3. ^ a b Edge MD; Green AR; Heathcliffe GR; et al. (1981). "Total synthesis of a human leukocyte interferon gene". Nature. 292 (5825): 756–62. Bibcode:1981Natur.292..756E. doi:10.1038/292756a0. PMID 6167861. {{cite journal}}: Unknown parameter |author-separator= ignored (help); Unknown parameter |month= ignored (help)
  4. ^ For example, the company DNA 2.0 was established in 2003 in Menlo Park, CA as a "synthetic genomics company" (quotated page).
  5. ^ "Difficult to Express Proteins". Sixth Annual PEGS Summit. Cambridge Healthtech Institute. 2010. Archived from the original on 11 May 2010. Retrieved 11 May 2010. {{cite web}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  6. ^ Liszewski, Kathy (1 May 2010). "New Tools Facilitate Protein Expression". Genetic Engineering & Biotechnology News. Bioprocessing. Vol. 30, no. 9. Mary Ann Liebert. pp. 1, 40–41. Archived from the original on 9 May 2010. Retrieved 11 May 2010Template:Inconsistent citations {{cite news}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)CS1 maint: postscript (link)
  7. ^ Welch M, Govindarajan M, Ness JE, Villalobos A, Gurney A, Minshull J, Gustafsson C (2009). Kudla, Grzegorz (ed.). "Design Parameters to Control Synthetic Gene Expression in Escherichia coli". PLoS ONE. 4 (9): e7002. Bibcode:2009PLoSO...4.7002W. doi:10.1371/journal.pone.0007002. PMC 2736378. PMID 19759823.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  8. ^ "Protein Expression". DNA2.0. Retrieved 11 May 2010.
  9. ^ a b Fuhrmann M, Oertel W, Hegemann P (1999). "A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii". Plant J. 19 (3): 353–61. doi:10.1046/j.1365-313X.1999.00526.x. PMID 10476082. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  10. ^ Mandecki W, Bolling TJ (1988). "FokI method of gene synthesis". Gene. 68 (1): 101–7. doi:10.1016/0378-1119(88)90603-8. PMID 3265397. {{cite journal}}: Unknown parameter |month= ignored (help)
  11. ^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene. 164 (1): 49–53. doi:10.1016/0378-1119(95)00511-4. PMID 7590320. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  12. ^ Gao X, Yo P, Keith A, Ragan TJ, Harris TK (2003). "Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences". Nucleic Acids Res. 31 (22): e143. doi:10.1093/nar/gng143. PMC 275580. PMID 14602936. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)
  13. ^ Young L, Dong Q (2004). "Two-step total gene synthesis method". Nucleic Acids Res. 32 (7): e59. doi:10.1093/nar/gnh058. PMC 407838. PMID 15087491.
  14. ^ Hillson NH, Rosengarten RD, Keasling JD (2012). "j5 DNA Assembly Design Automation Software". ACS Synthetic Biology. 1 (1): 14–21. doi:10.1021/sb2000116.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  15. ^ Hoover DM, Lubkowski J (2002). "DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis". Nucleic Acids Res. 30 (10): e43. doi:10.1093/nar/30.10.e43. PMC 115297. PMID 12000848. {{cite journal}}: Unknown parameter |month= ignored (help)
  16. ^ Villalobos A, Ness JE, Gustafsson C, Minshull J, Govindarajan S (2006). "Gene Designer: a synthetic biology tool for constructing artificial DNA segments". BMC Bioinformatics. 7: 285. doi:10.1186/1471-2105-7-285. PMC 1523223. PMID 16756672.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  17. ^ Tian J; Gong H; Sheng N; et al. (2004). "Accurate multiplex gene synthesis from programmable DNA microchips". Nature. 432 (7020): 1050–4. Bibcode:2004Natur.432.1050T. doi:10.1038/nature03151. PMID 15616567. {{cite journal}}: Unknown parameter |author-separator= ignored (help); Unknown parameter |month= ignored (help)
  18. ^ Matzas M; et al. (2010). "High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing". Nature Biotechnology. 28: 1291–1294. doi:10.1038/nbt.1710. PMID 21113166. {{cite journal}}: Explicit use of et al. in: |author= (help)
  19. ^ Schwartz JJ, Lee C, Shendure J. (2012). "Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules". Nature Methods. 9: 913–915. doi:10.1038/nmeth.2137. PMID 22886093.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  20. ^ "Genome Transplantation in Bacteria: Changing One Species to Another". Science. 2007-06-28. Archived from the original on 24 May 2010. Retrieved 2010-05-22. {{cite web}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  21. ^ Pilkington, Ed (2009-10-06). "I am creating artificial life, declares US gene pioneer". London: The Guardian. Archived from the original on 28 May 2010. Retrieved 2010-05-22. {{cite news}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  22. ^ "Synthetic Genome Brings New Life to Bacterium" (PDF). Science. 2010-05-21. Archived from the original (PDF) on 25 May 2010. Retrieved 2010-05-21. {{cite web}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  23. ^ "How scientists made 'artificial life'". BBC News. 2010-05-20. Archived from the original on 23 May 2010. Retrieved 2010-05-21. {{cite news}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  24. ^ Langreth, Robert (April 19, 2012). "Artificial DNA Can Replicate in Lab, Researchers Find". Bloomberg. Retrieved May 8, 2012.
  25. ^ Pinheiro, Vitor; Taylor, Alexander; McLaughlin, Christopher; Abramov, Mikhail; Renders, Marleen; Zhang, Su; Chaput, John; Wengel, Jesper; Peak-Chew, Sew-Yeu; Herdewijn, Piet; Holliger, Philipp (2012). "Synthetic Genetic Polymers Capable of Heredity and Evolution". Science. 336 (6079). AAAS: 341–344. Bibcode:2012Sci...336..341P. doi:10.1126/science.1217622. Retrieved May 8, 2012. {{cite journal}}: |first10= missing |last10= (help); Unknown parameter |unused_data= ignored (help)

References

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