| The application of nucleic acid chemistry to studies on the functional organization of gene control regions. | |
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MedLine Citation:
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PMID: 6300026 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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A new, extremely high yield method for synthesizing deoxyoligonucleotides has been developed. Syntheses are completed on a silica gel polymer support using appropriately protected deoxymononucleotide 3'-phosphoramidites. These mononucleotide intermediates are synthesized using simple organo chemical procedures and can be stored indefinitely as dry powders. Activation is achieved by adding a weak acid such as tetrazole to an acetonitrile solution of the mononucleotide. Chemical steps for the addition of one nucleotide to the support are the following: (1) detritylation using ZnBr2 in nitromethane/methanol (5 min); (2) condensation of a 5'-O-dimethoxytrityldeoxynucleoside 3'-methoxy-N, N-dimethylamino phosphine with the support bound nucleoside (5 min); (3) blocking unreacted, support bound nucleoside hydroxyl groups with acetic anhydride (5 min); (4) oxidation of the phosphite to the phosphate with I2 (2 min). Completed deoxyoligonucleotides are isolated by first sequentially treating with thiophenol, t-butylamine, ammonium hydroxide, and 80% acetic acid and then purifying to homogeneity by electrophoresis on polyacrylamide gels. The method is extremely fast (approximately 30 min per cycle), yields in excess of 98% per condensation are obtained, and isolation of the final product is a simple, one step purification. This methodology has been used to synthesize several lac operators containing altered sequences. The results of binding studies indicate that lac repressor does not recognize a unique DNA sequence. |
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Authors:
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M H Caruthers |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Princess Takamatsu symposia Volume: 12 ISSN: - ISO Abbreviation: Int. Symp. Princess Takamatsu Cancer Res. Fund Publication Date: 1982 |
Date Detail:
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Created Date: 1983-05-05 Completed Date: 1983-05-05 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 9301172 Medline TA: Princess Takamatsu Symp Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 295-306 Citation Subset: IM |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Base Sequence DNA / genetics* Genes* Indicators and Reagents Methods Oligodeoxyribonucleotides / chemical synthesis* Oligonucleotides / chemical synthesis* Silicon Dioxide |
| Grant Support | |
ID/Acronym/Agency:
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1 KO4 GM00076/GM/NIGMS NIH HHS; GM21120/GM/NIGMS NIH HHS; GM25680/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Indicators and Reagents; 0/Oligodeoxyribonucleotides; 0/Oligonucleotides; 60650-90-0/silochrome; 7631-86-9/Silicon Dioxide; 9007-49-2/DNA |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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