Laboratory Synthesis of Oligonucleotides

The laboratory synthesis of oligonucleotides and polynucleotide fragments is a subject of great importance. Much has already been acheived (Chapter 10.4C). In addition to hydrolysis, however, the nucleic acids are very sensitive to a wide range of chemical reactions, for example, the heterocyclic bases are subject to alkylation, oxidation and reduction. Generally only mild reactions can be used in the construction of an oligonucleotide chain... 

The triphenylmethyl, or trityl, ether (-CPha, or Tr) can be cleaved by reduction or with acid, via the stabilized trityl carbocation. Derivatives of the trityl group are widely used as protective groups in the laboratory synthesis of oligonucleotides (DNA). 

This introduction, and the examples of synthetic procedures given below, are limited to presentation of methods developed in the authors laboratory space limitation does not allow the presentation of results of all research groups contributing to the synthesis of oligonucleotide analogs of a defined sense of chirality at phosphorus. These contributions are acknowledged by pertinent citations. 

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

The automated chemical synthesis of moderately long oligonucleotides (about 100 nucleotides) of precise sequence is now a routine laboratory procedure. Each synthetic cycle takes but a few minutes, so an entire molecule can be made by synthesizing relatively short segments that can then be ligated to one another. Oligonucleotides are now indispensable for DNA se-... 

Simultaneously, numerous academic groups started and even reoriented their activities towards the implantation of solid-phase strategies in their laboratories. Although progress towards fulfillment of the initial great expectations has slowed in recent years, solid-supported chemistry is now a very useful tool in all modern organic chemistry laboratories [11] and, of course, is the method of choice for both research and industrial synthesis of peptides [12] and oligonucleotides [13]. 

Synthesis on solid supports was first developed by Merrifield [1] for the assembly of peptides. It has expanded to include many different applications including oligonucleotide, carbohydrate, and small-molecule assembly (see Chapters 11 and 14). The repetitive cycle of steps involved in the solid-phase synthesis of biopolymers can be performed manually using simple laboratory equipment or fully automated with sophisticated instrumentation. This chapter examines typical solid-phase reaction kinetics to identify factors that can improve the efficiency of both manual and automated synthesis. The hardware and software features of automated solid-phase instruments are also discussed. The focus of this discussion is not on particular commercial model synthesizers but on the basic principles of instrument operation. These considerations can assist in the design, purchase, or use of automated equipment for solid-phase synthesis. Most contrasting features have advantages and disadvantages and the proper choice of instrumentation depends on the synthetic needs of the user. 

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