Precise discrimination among homologous sequences by DNA probe column chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Oligonucleotide probes are crucial for DNA sequence detection.
• High-performance liquid chromatography (HPLC) offers high resolution for separation.
• Developing sensitive methods for distinguishing homologous DNA sequences is essential.

Purpose of the Study:

• To investigate the use of immobilized oligonucleotide probes in HPLC for discriminating homologous DNA sequences.
• To evaluate the impact of sequence mismatches on chromatographic profiles.
• To explore the relationship between hybrid stability and elution profiles.

Main Methods:

• Synthesizing and immobilizing oligonucleotide probes onto HPLC gel matrix.
• Packing the functionalized gel into HPLC columns.
• Performing high-performance liquid affinity chromatography (HPLAC) under isothermal and isocratic conditions.
• Analyzing chromatograms of perfectly matched and single-mismatch oligonucleotide samples.

Main Results:

• HPLAC successfully discriminated between perfectly matched and single-mismatch sequences.
• Chromatographic profiles varied significantly with column temperature and mismatch type.
• Elution profile deformation correlated with the stability of the DNA hybrid formed between the sample and immobilized probe.

Conclusions:

• Immobilized oligonucleotide probes in HPLAC provide a sensitive method for detecting sequence variations.
• The stability of the probe-target hybrid is a key determinant of chromatographic separation.
• This approach holds potential for applications in genetic analysis and diagnostics.