Separation type: Bridge Ion Separation Technology, or BIST™ by SIELC Technologies
HPLC Method for Analysis of DNA Oligo, 5 bases (AAAAA) on BIST AC Column by SIELC Technologies
High Performance Liquid Chromatography (HPLC) Method for Analysis of DNA Oligo, 5 bases (AAAAA)
Synthesis of DNA Oligonucleotide (AAAAA)
- Automated Synthesis:
- DNA oligonucleotides, including a sequence like “AAAAA”, are typically synthesized using automated synthesizers based on solid-phase synthesis.
- The process involves the sequential addition of nucleotide residues to the growing chain in a step-wise manner, starting from the 3′-end of the oligo.
- Chemistry:
- The synthesis uses phosphoramidite chemistry, where each nucleotide to be added is in its protected phosphoramidite form.
- The process includes coupling, capping, oxidation, and deprotection steps.
- Purification:
- After synthesis, the oligonucleotide is usually cleaved from the solid support and deprotected.
- It is then purified, commonly by methods like HPLC or PAGE (polyacrylamide gel electrophoresis), depending on the required purity level.
Using DNA Oligo (AAAAA)
The application of a DNA oligo with a sequence “AAAAA” can vary based on the context:
- Research and Studies:
- Molecular Biology: In molecular biology, such oligos can be used as primers for PCR, sequencing, or as probes in hybridization experiments.
- Binding Studies: The poly(A) sequence may be used to study binding interactions with proteins, such as DNA-binding proteins or enzymes.
- Medical and Diagnostic Applications:
- As part of diagnostic kits, especially in assays that require hybridization to a complementary sequence.
- Educational Purposes:
- Demonstrating basic principles of nucleic acid chemistry and genetics.
- Nanotechnology:
- In DNA nanotechnology, specific sequences of DNA are used to form structures and shapes at the nanoscale. A poly(A) sequence might be part of a larger structure.
Using SIELC’s newly introduced BIST™ method, this oligonucleotide can be retained on a negatively-charged, cation-exchange BIST™ A column. There are two keys to this retention method: 1) a multi-charged, positive buffer, such as TMEDA formate, which acts as a bridge, linking the negatively charged dye to the negatively-charged column surface and 2) a mobile phase consisting mostly of organic solvent (such as MeCN) to minimize the formation of a solvation layer around the charged analytes. Using this new and unique analysis method, oligonucleotide can be separated, retained, and detected at 260 nm.
Please read more on oligonucleotides analysis by HPLC in our April’s 2023 newsletter.
Condition
| Column | BIST AC, 4.6 x 100 mm, 5 µm, 100 A |
| Mobile Phase | MeCN – 60% |
| Buffer | TMEDA Formate pH 4.0 – 20 mM |
| Flow Rate | 1.0 ml/min |
| Detection | UV 260 nm |
| Sample | 0.021 mg/ml in EtOH/H2O – 50/50% |
| Injection volume | 1 µl |
| LOD* | 15 ppb |
Description
| Class of Compounds | Oligonucleotides |
| Analyzing Compounds | Oligonucleotides |
Application Column
