Rapid synthesis of oligodeoxyribonucleotides IV. Improved solid phase synthesis of oligodeoxy-ribonucleotides through phosphotriester intermediates

Abstract

A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.

Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter

“…Attempts to purify the oligomer mixtures obtained from the syntheses with HPLC and ion-exchange columns using published elution buffer systems (29,30) did not give satisfactory results in our hands. It has been observed that oligomers containing several G residues in a row could not be separated (28).…”

The use of synthetic oligodeoxynucleotide primers in cloning and sequencing segment 8 of influenza virus (A/PR/8/34)

“…The dodecanucleotide d(A-G-C-A-A-A-A-G-C-A-G-G) was chemically synthesized by the solid phase phosphotriester method on a polydimethylacrylamide support as described previously [9,10]. In the synthesis of the tridecanucleotide d(A-G-T-A-G-A-A-A-C-A-A-G-G) the following significant improvements were made: (1) Acidic removal of terminal 5'-0-dimethoxytrityl protecting groups was effected by 2 x 2 min treatments with 101/o trichloroacetic acid in chloroform.…”

“…(2) The coupling agent, mesitylenesulphonyl-3-nitro-1,2,4-triazole, was used in coupling reactions instead of the previously used triisopropylbenzenesulphonyl tetrazole owing to greater stability and faster coupling rate [11]. The tridecanucleotide was purified by ion exchange and reverse phase high performance liquid chromatography as described previously [9,10]. Preparation of dsDNA DNA for the M13 shotgun was prepared as described previously [8].…”

The construction of a syntheticEscherichia coli trppromoter and its use in the expression of a synthetic interferon gene

“…A more likely explanation for our results may be that pAT153, the vector used in our studies, has a 2-3 fold higher copy number than its parent plasmid pER322, which formed the basis of the other expression systems. This difference might result in the number of trp operator sequences present in a cell carrying a plasmid such as pSTP2 exceeding the maximum number of trp repressor molecules (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) which can be produced from a single R gene (36 …”

“…Oligonucleotides were synthesised by solid-phase phosphotriester methodology and purified as described previously (28,29), except that in Partisil 10-SAX ion exchange hplc 30% formamide was substituted for aqueous ethanol in elution buffers (30). This gives good resolution with lower phosphate concentrations at room temperature and extends column life.…”