High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Accurate base composition analysis is crucial for synthetic nucleic acids.
• Existing methods may face limitations with modified bases or complex samples.
• High-performance liquid chromatography (HPLC) is a standard for nucleoside separation.

Purpose of the Study:

• To present an improved, highly accurate, and sensitive method for determining the base composition of synthetic oligodeoxyribonucleotides.
• To validate the method's applicability to various lengths of oligodeoxyribonucleotides and modified bases.
• To assess the method's potential for analyzing high-molecular-weight genomic DNA.

Main Methods:

• Enzymatic digestion of synthetic oligodeoxyribonucleotides to yield nucleosides.
• High-resolution High-Performance Liquid Chromatography (HPLC) separation of the resulting nucleosides.
• Quantification of nucleosides to determine empirical base composition.

Main Results:

• Demonstrated excellent agreement between experimental and theoretical base compositions for oligodeoxyribonucleotides (18-150 bases).
• The enzymatic digestion was quantitative and unaffected by common base modifications.
• The method successfully assessed the degree of base deprotection and chemical modification.

Conclusions:

• The presented method offers a significant improvement for base composition analysis of synthetic oligodeoxyribonucleotides.
• The technique is robust, sensitive, and applicable to modified bases and genomic DNA.
• This method provides a reliable tool for quality control in oligonucleotide synthesis and analysis.