Gene synthesis
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Gene synthesis is the process of synthesizing an artificially designed gene into a physical DNA sequence.
Gene synthesis was first demonstrated by Har Gobind Khorana in 1970 for a short artificial gene. Nowadays, commercial gene synthesis services are available from hundreds of companies worldwide, with a price often below $1 a base pair. Some expressed concern that such services could be used to create new strains of existing viruses/bacterias, or to resurrect extinct biological hazard organisms. Gene synthesis is a method in molecular biology, comprising the complete de novo production of structural genes, employing a combination of organic chemistry and molecular biology procedures, without having biological templates in hand. It has become an important tool in many fields of recombinant DNA technology including heterologous gene expression, vaccine development, gene therapy and molecular engineering. Frequently the synthesis of nucleic acid sequences is more economical than classical cloning and mutagenesis procedures.
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[edit] 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. However, 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. 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 redundancy 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 can have a dramatic effects. Further optimizations such as removing RNA secondary structures can also be included. Computer programs are written to perform these and other simultaneous optimizations are used to handle the enormous complexity of the task. 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.
[edit] Standard Methods
[edit] 1 ) 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 phophoramidite 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. 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.
[edit] 2) 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 (4). 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 (4), thermodynamically balanced inside-out (TBIO) PCR (5) or combined approaches (6).
[edit] 3) 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 (7, 8).
[edit] 4) Error correction procedures
To overcome problems associated with oligonucleotide quality several elaborate strategies have been developed, employing either separately prepared fishing oligonucleotides (9), mismatch binding enzymes of the mutS family (10) or specific endonucleases from bacteria or phages (11). Nevertheless, all these strategies increase time and costs for gene synthesis based on the annealing of chemically synthesized oligonucleotides.
[edit] Gene synthesis market
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. Active gene synthesis providers in the market are ShineGene,GENEART, DNA2.0., Epoch Biolabs Inc., GenScript, BioBasic, and Biomatik.
[edit] See also
[edit] References
(1) Gupta,N.K., Ohtsuka,E., Sgaramella,V., Buchi,H., Kumar,A., Weber,H. and Khorana,H.G. (1968) Studies on polynucleotides, 88. Enzymatic joining of chemically synthesized segments corresponding to the gene for alanine-tRNA. Proc. Natl Acad. Sci. USA, 60, 1338–1344. Click here to read
(2) Fuhrmann M, Oertel W, Hegemann P. A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J. 1999 Aug;19(3):353-61. Click here to read
(3) Mandecki,W. and Bolling,T.J. (1988) FokI method of gene synthesis. Gene, 68, 101–107. Click here to read Abstract
(4) Stemmer,W.P., Crameri,A., Ha,K.D., Brennan,T.M. and Heyneker,H.L. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164, 49–53. Click here to read Abstract
(5) Gao X, Yo P, Keith A, Ragan TJ, Harris TK. 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. 2003 Nov 15;31(22):e143. TBIO-PCR
(6) Young L, Dong Q. Two-step total gene synthesis method. Nucleic Acids Res. 2004 Apr 15;32(7):e59. Click here to read
(7) Hoover,D.M. and Lubkowski,J. (2002) DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res., 30, e43. Click here to read
(8) Villalobos A, Ness JE, Gustafsson C, Minshull J, Govindarajan S. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. BMC Bioinformatics. 2006 Jun 6;7:285. Click here to read
(9) Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, Church G. Accurate multiplex gene synthesis from programmable DNA microchips. Nature. 2004 Dec 23;432(7020):1050-4. Click here to read Abstract
(10) Carr PA, Park JS, Lee YJ, Yu T, Zhang S, Jacobson JM. Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. 2004 Nov 23;32(20):e162. Click here to read
(11) Fuhrmann M, Oertel W, Berthold P, Hegemann P. Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage. Nucleic Acids Res. 2005 Mar 30;33(6):e58. Click here to read
[edit] External links
- Biodirectory
- Rewriting the genetic code - an Instant Insight discussing how researchers' dreams of automated gene synthesis could soon become a reality from the Royal Society of Chemistry
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