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. 1994 Aug 11;22(15):2998–3004. doi: 10.1093/nar/22.15.2998

Reagents for the preparation of two oligonucleotides per synthesis (TOPS).

P M Hardy 1, D Holland 1, S Scott 1, A J Garman 1, C R Newton 1, M J McLean 1
PMCID: PMC310267  PMID: 8065912

Abstract

In order to increase the efficiency of use of automated DNA synthesizers (i.e. the number of oligomers prepared per day), we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotides sequence upon the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over.

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Selected References

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  1. Gait M. J. DNA/RNA synthesis and labelling. Curr Opin Biotechnol. 1991 Feb;2(1):61–68. doi: 10.1016/0958-1669(91)90062-a. [DOI] [PubMed] [Google Scholar]
  2. Gish G., Eckstein F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science. 1988 Jun 10;240(4858):1520–1522. doi: 10.1126/science.2453926. [DOI] [PubMed] [Google Scholar]
  3. Mag M., Engels J. W. Synthesis and selective cleavage of oligodeoxyribonucleotides containing non-chiral internucleotide phosphoramidate linkages. Nucleic Acids Res. 1989 Aug 11;17(15):5973–5988. doi: 10.1093/nar/17.15.5973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  5. McCafferty J., Jackson R. H., Chiswell D. J. Phage-enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage. Protein Eng. 1991 Dec;4(8):955–961. doi: 10.1093/protein/4.8.955. [DOI] [PubMed] [Google Scholar]
  6. Newton C. R., Kalsheker N., Graham A., Powell S., Gammack A., Riley J., Markham A. F. Diagnosis of alpha 1-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products. Nucleic Acids Res. 1988 Sep 12;16(17):8233–8243. doi: 10.1093/nar/16.17.8233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  8. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. van Wezenbeek P. M., Hulsebos T. J., Schoenmakers J. G. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene. 1980 Oct;11(1-2):129–148. doi: 10.1016/0378-1119(80)90093-1. [DOI] [PubMed] [Google Scholar]

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