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Concentration and extinction coefficient determination for oligonucleotides and analogs using a general phosphate
1Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland, 21205, USA.
Analytical Biochemistry
|September 5, 1996
Summary
A novel general phosphate analysis (GPA) quantifies nucleic acid oligomers and analogs by measuring phosphorus content. This method accurately determines concentrations for various backbone linkages and base analogs.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Molecular Biology
Background:
- Accurate quantification of nucleic acid oligomers is crucial for molecular biology and drug development.
- Existing methods may have limitations in analyzing diverse backbone linkages or base analogs.
Purpose of the Study:
- To develop a versatile and accurate method for quantifying nucleic acid oligomers and their analogs.
- To establish a general phosphate analysis (GPA) applicable to various chemical modifications.
Main Methods:
- Developed a general phosphate analysis (GPA) based on stoichiometric phosphorus determination.
- Employed high-temperature acid digestion to convert oligomers to orthophosphate.
- Utilized colorimetric analysis via phosphomolybdate complex formation for quantification.
Main Results:
- GPA successfully quantified oligomers with phosphodiester, methylphosphonate, and phosphorothioate backbones.
- Extinction coefficients were determined for different backbone types.
- The method demonstrated accuracy comparable to existing quantitative and predictive techniques.
Conclusions:
- GPA provides a robust method for analyzing nucleic acid oligomers and analogs.
- The technique is applicable to modified nucleosides and diverse backbone chemistries.
- GPA offers a valuable tool for researchers working with synthetic oligonucleotides.
