Synthesis of Oligonucleotides and Polynucleotides

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... 

Chemical Synthesis of Oligonucleotides and Polynucleotides of Defined Sequences.—... 

Reaction conditions have been described for the synthesis of oligonucleotides by the use of polynucleotide phosphorylase, which uses a dinucleoside (3 -50-nionophosphate as primer and nucleoside 5 -phos-phates. The enzyme also used as primers unnatural phosphodiesters, such as nitrophenylated nucleoside 5 -phosphates. The yields of these reactions, however, are considerably lower as compared to reactions with natural phosphodiesters as primer. The yields of the reaction were found to be relatively insensitive to variations in salt concentration or in pH of the incubation mixtures. Optimal salt conditions, i.e., mM MgCh and 0.4-1,0 MNaCl, are similar to those that are used in oligonucleotide synthesis with normal primers. However, it is advisable that incubation mixtures not exceed volumes of 1 ml. In our experience, it is better to split larger volumes into smaller ones, if large quantities of oligonucleotide are to be synthesized. 

Compelling—though indirect—evidence for this view comes from model studies of possible prebiotic template-directed oligonucleotide synthesis. In these experiments, a single-stranded homopurine or homopyrimidine polynucleotide is used as a template, and the ability of the prebiotic system to achieve synthesis of the complementary strand from mononucleotide precursors is measured. Since there is no polymerase present, the success of the system depends on the ability of the mononu-... 

For mutagenesis to introduce the stop codons, the protocols in Subheadings Oligonucleotide Phosphorylation with T4 Polynucleotide Kinase, Annealing of the Oligonucleotide to the Template, and step 1 of Enzymatic Synthesis of CCC-dsDNA can be scaled down by a factor of ten. The reaction product can be used directly to transform E. coli using any standard procedure. 

Polynucleotides. Aryl phosphorodichloridates (1,847) are useful for synthesis of short oligonucleotides by the phosphotriester procedure however, the reactions are slow and yields are low as the chain length is increased. However, aryl phosphorodichloridites react with hydroxyl groups in THF at -78°... 

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