Methods in molecular biology and gene technology

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Methods in molecular biology and gene technology ..

27/06/2016 · Oligonucleotide Synthesis Methods and Applications Methods in Molecular Biology Lloyd Butts

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Gene Synthesis - DNA Synthesis - from .11/bp - …

Relatively high molecular weight DNA sequences have been prepared successfully by the phosphotriester approach in solution by following essentially the procedure indicated in outline in Figure 1a. However, solution-phase synthesis is relatively laborious in that chromatographic purification steps are usually necessary after each coupling step. Nevertheless, if a very large quantity of a specific sequence is required (see text below), solution-phase synthesis may very well prove to be the method of choice. If, on the other hand, relatively small (i.e., milligram to gram) quantities of material are required for biological or biophysical studies, there is little doubt that solid-phase synthesis is to be preferred. While all three of the above phosphorylation methods (Fig. 1) have been used in solid-phase synthesis, the phosphoramidite approach (9) has emerged as the method of choice. This is mainly because its use leads to high coupling efficiencies and no significant side reactions. Furthermore, most commercial automatic synthesizers have been designed specifically to accommodate phosphoramidite chemistry. The main advantages of solid-phase synthesis, particularly by the phosphoramidite approach, are: (1) that it is very rapid and a DNA sequence containing, say, 50 nucleotide residues can easily be assembled and unblocked within one day; (2) only one purification step is required at the end of a synthesis as the growing DNA sequence is attached to a solid support (such as controlled pore glass [CPG] or polystyrene), and the excesses of all reagents are washed away; (3) all chemical reactions can be made to proceed in very high yield by using large excesses of reagents; and (4) the whole process may be fully automated in a DNA synthesizer. Solid-phase DNA synthesis has been developed to such an extent that the whole process can be carried out by a competent technician with no specialist knowledge of nucleotide chemistry. Automatic synthesizers, some of which are capable of assembling several different specific DNA sequences simultaneously, are readily available, and all the necessary building blocks [particularly phosphoramidites 17] and other reagents and solvents may be purchased in containers that are designed to be attached directly to the synthesizer.

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Why gene synthesis?
Gene synthesis can artificially synthesize double-stranded DNA in vitro, with an assembly capacity of 50bp to 12Kb products. Gene synthesis differs from traditional molecular cloning and PCR cloning in several ways. The traditional molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombined DNA molecules and to direct their replication within host organisms. However, not every gene has high-efficiency expression in these systems, meaning that molecular cloning may not be the best option for these genes. Instead, through gene synthesis, it is possible to avoid this problem by creating a new system with high-efficiency expression of the target gene.

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Synthesis, Gene Silencing, and Molecular Modeling Studies of 4′-C-Aminomethyl-2′-O-methyl Modified Small Interfering RNAs.

Of the three phosphorylation methods described above, only the phosphotriester approach (Fig. 1a) is really suitable for the synthesis of DNA sequences in solution. This method, which was developed largely in the 1970s, is very versatile and is particularly suitable for the coupling of oligonucleotide blocks (i.e., the addition of two or more nucleotide residues at a time) as well as for stepwise synthesis. Phosphotriester block coupling was a key feature of the original synthesis of the human insulin gene (12). Although the methodology has been refined (13) since then, the development of automated solid-phase synthesis (see above) in the 1980s provided a much faster and less labor-intensive method for the preparation of the very small (usually milligram or even smaller) quantities of synthetic DNA sequences that are generally required in molecular biology. Solution-phase synthesis is much more laborious in that it is normally advisable to purify the products by chromatography after each coupling step. Although such purification processes need not necessarily amount to much more than filtration through a bed of silica gel, they are time consuming. Furthermore, solution-phase synthesis has not yet been automated. It is, nevertheless, not at all unlikely that solution-phase synthesis will become the method of choice if really large (i.e., multikilogram to tonne) quantities of moderately sized (containing ca. 20 nucleotide residues) DNA sequences or their analogs are required in anti-sense or antigene chemotherapy. Automated solid-phase synthesis has recently been scaled-up to the multigram level (14) in order to provide sufficient material for clinical trials. However, if such clinical trials are successful and very much larger quantities of pure DNA sequences and their analogs are required for drug purposes, further substantial scaling-up of solid-phase synthesis may not prove to be a practical proposition. It is quite likely that the solution-phase synthesis or perhaps a combination of solution-phase and solid-phase synthesis might lend itself much more readily to scaling-up. The phosphotriester approach has the further advantage that the fully-protected intermediates obtained are soluble in organic solvents and may, therefore, be purified by conventional chromatographic techniques, and, after all of the protecting groups have been removed, the unprotected DNA sequences obtained may, if necessary, be further purified in the same way as material that has been prepared on a solid support.

The introduction of methods for the chemical synthesis of oligo- and poly- deoxyribonucleotides (DNA sequences) has had a very considerable effect on the development of molecular biology. This is clearly apparent from other sections of the topic. The three most important factors to be taken into account in the chemical synthesis of DNA sequences are:(1) the choice of suitable protecting groups for the 2′-deoxyribonucleoside building blocks [1], (2) the development of phosphorylation procedures that are suitable for the introduction of the internucleotide linkages, and (3) the purification of the synthetic DNA sequences themselves. The choice of protecting groups is of crucial importance. The protecting groups selected should be easy to introduce; they should also remain completely intact throughout the assembly of the DNA sequences and be easily removable under conditions where the synthetic DNA is completely stable.

Molecular Biology and Biotechnology - iMedPub

14/01/2018 · DNA Synthesis (Molecular Biology) The introduction of methods for the chemical synthesis of oligo ..

N2 - DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.

DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.

Gene Synthesis: Methods and Protocols (Methods in Molecular Gene Synthesis: Methods and Protocols (Methods in Molecular Biology, Vol
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AB - DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.

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