Artificial gene synthesis
Artificial gene synthesis is a method of synthetic biology, which is used to create artificial genes in the laboratory. Based on oligonucleotide synthesis, it differs in that of the molecular cloning and polymerase chain reaction (PCR), that the user does not pre-existing DNA needed. Thus, it is possible a complete, double-stranded DNA molecule, with no restrictions in sequence or length, to manufacture. The method was used to design operational, bacterial chromosomes, which contained approximately one million base pairs, to produce.
The first synthesis of the entire gene, a yeast tRNA, was accomplished by Har Gobind Khorana and his colleagues in 1972. The syntheses of the first peptide- or protein-coding gene were each carried out in the laboratories of Herbert Boyer and Alexander Markham.
Commercial Gensyntheseaufträge are now handled by numerous companies worldwide, with some who have been specially set to this branch of genetics. The current approach of gene synthesis is usually a combination of organic chemistry and molecular biology techniques, it may be that all genes " de novo ", can be synthesized without existing DNA template. Gene synthesis is in many fields of recombinative DNA technology has become an important tool. The synthesis of nucleotide bases is often more economical than classical cloning or mutation methods.
Gene optimization
Because of the potential to produce long stretches of DNA accurately and for ever lower prices causing increasingly more and more demand on the Gensynthesefeld, more and more attention is paid to the adaptation of the genes for specific purposes. In the early days of genome sequencing, gene synthesis was used as an expensive source of cDNA. This was obtained from genomic DNA or partial cDNA has been difficult to clone. This method was as higher quality sources for cDNA arose no longer mandatory
Large amounts of proteins from naturally occurring gene sequences, or at least the protein-coding region of the gene; the open reading frame to win, can often be difficult. This is a problem which content of various scientific conferences was. Many of the needed by molecular biologists proteins are normally regulated so that they are only very slightly expressed in wild-type cells. By fitting design of these genes can improve gene expression in many cases. Due to the fault tolerance, the rewriting of the ORF is possible under certain conditions. It is possible to change to one-third of base pairs, are still the same protein is produced. The possible number of alternative designs of the DNA sequence for a particular protein is astronomical. For a protein sequence of 300 amino acids, there are over 10150 codon combinations that would produce an identical protein. Optimization methods, such as exchanging hardly used codons with more common, sometimes have a dramatic effect. Furthermore, even optimizations such as, the removal of secondary structures are used. In the case of E. coli, is finally protein expression by overriding the use of codons found within amino acids include tRNA wärent undersupply be stored is maximized. To cope with the complexity of the various simultaneous optimization computer programs are now used. A well- optimized gene can enhance protein expression by a factor of 2 to 10. In some cases, improvements by a factor of 100 are recorded. Due to the large number of modified nucleotides, is the only appropriate way is rewritten genes to create the gene synthesis.
Standard methods
Chemical synthesis of oligonucleotides
Oligonucleotides can be chemically synthesized, Nukloeosid phosphoramidites are reacted with each other by a phosphoramidite synthesis. These blocks are initially protected from, ie their amines, hydroxyl groups, and phosphate groups the protective groups are attached, which do not react during the oligonucleotide synthesis, and are afterwards removed. In each step of the synthesis, however, the next respective 5'-hydroxy group of the product is deprotected in order for the next phosphoramidite was added and a new base can accumulate. The chain grows from 3 'to 5' end, which is exactly reversed for biosynthesis.
Since it is chemical processes, the yield of the oligonucleotides is reduced to the correct sequences of the sequence length. A small error probability in each synthesis step adds up to inevitably. Thus, this technique is more suited to the production of short sequences. The current limit for oligonucleotides with sufficient quality to be used directly for biological processes, are 200 bp. By means of HPLC, the synthetic product are cleaned of false sequences.
When a large number of different oligonucleotides synthesized simultaneously a carrier material (eg glass ), is called the product " gene chip ".
Annealing of oligonucleotides
Normally, a set of individually designter oligonucleotide is established via automated solid phase synthesizer and then purified and then connected by specific annealing and ligation or polymerase reaction. To enhance the annealing of the oligonucleotides, the synthesis step is based on a combination of thermostable DNA ligase, and a polymerase enzyme. There are now described a variety of methods of gene synthesis. Examples include the ligation of phosphorylated overlapping oligonucleotides, the Fok I and an adapted form of the ligase chain reaction for gene synthesis. In addition, some PCR assembly were described approaches. They typically use oligonucleotides with a length of 40-50 bp overlap with each other. These oligonucleotides are designed such that they together cover the major part of the sequence of both strands. The complete molecule is then gradually over overlap extension PCR ( OE), through TBIO PCR or combined methods, made . The usual size of synthesized genes has a length of 600-1200 bp, although already much longer genes were generated by ligation of less than 1,000 bp long parts. In this scale, it is necessary to test for the individual parts each several possible clones based automated sequencing methods.
Limitations
Moreover, since the generation of the complete gene, the efficient and the precise arrangement of long, single-stranded oligonucleotides is Abhäng, there are some critical parameters for the success of the synthesis: larger sequence regions with secondary structures, which are caused by trapped repetitions; exceptionally high or low GC content; repetitive structures. Normally, these segments of a gene that can only be generated by dividing a number of small parts and subsequently assembling the individual parts. This in turn leads to significant increase in the time and effort.
The result of a gene synthesis is highly dependent on the quality of the oligonucleotides were used as. For this annealing based approach, the oligonucleotides act directly and exponentially on the accuracy of the product. Alternatively needs after oligos lower quality were merged by gene synthesis, more effort will be operated subsequently to ensure the quality of the gene. This is usually done by Standardklonieren followed by transformation and analysis of the clones by sequencing. However, this is a time consuming process.
Another problem encountered with the conventional gene synthesis, is the frequent occurrence of sequence errors, due to the use of chemically synthesized oligonucleotides. As a result of this, the percentage of correct products drops sharply with increasing number of used oligos.
The mutation problem can solved by warden shorter the oligonucleotides as building blocks of the gene. However, all require Assemblemethoden that the primers are combined in one vessel. Thus, short overhangs are not always with their complementary primers anneal precisely and accurately, which in turn affects the formation of the complete gene.
Manually create oligonucleotides is a laboratory practice and not necessarily guaranteed the successful synthesis of the desired gene. For optimal results, almost all annealing, the melting temperature of the overlapping regions for all oligonucleotides must be similar. The necessary primer optimization should be performed using a specialized oligonucleotide design programs. Here already some solutions were found automated primer design for gene synthesis.
Fehlerkorigierende method
To resolve the problems caused by poor Oligonukleotidqualtität, several strategies have been developed. There are either specially prepared Fishingoligonukleotideemploying, Mismatchbindingenzyme the mutS family or specific endonuclease from bacteria or phages used. Nonetheless, all of these strategies cost time and money.
Also is partially parallel sequencing large Oligobibliotheken as a means, used to find matching molecules. In one method, oligonucleotides are sequenced on a 454 Pyrosequenzierplattform and a robot system maps the individual beads and selects the matching the sequence.
Increasingly, even whole sets of genes in demand, with mutually similar sequences or with different sequences that have only a few base pair differences. Almost all of the therapeutic proteins in the development of such monoclonal antibodies, can be optimized by testing of numerous genetic variants for improved function or expression.