Analysis of Oligonucleotides by Capillary Gel Electrophoresis with the Agilent 7100 Capillary Electrophoresis System

Significance of the topic

Capillary gel electrophoresis (CGE) is a powerful method to resolve oligonucleotides by size, overcoming the limitations of capillary zone electrophoresis in which these analytes display similar mobilities. This high-resolution approach is critical in research, quality control and therapeutic oligonucleotide development, where distinguishing molecules differing by a single nucleotide is essential.

Study objectives and overview

This work aimed to systematically evaluate experimental parameters on the Agilent 7100 CE system for the separation of four oligonucleotide standards. The study assessed:

• Different sieving gels composed of polyethylene glycol (PEG) with or without acetonitrile
• Detection filters at 260 nm and 280 nm
• Neutral-coated capillaries (PVA, µSIL WAX, µSIL DNA)
• Method variables such as temperature, voltage and detection geometry

Methodology and instrumention

Reagents included 200 mM BisTris/boric acid buffer and PEG-based gels prepared at 27% polymer content, with one variant containing 20% acetonitrile. Three capillary types with 75–100 µm inner diameters were conditioned via automated washing steps. Sample injection employed a combination of pressure and electrokinetic loading, with detection using a UV diode array at 260 nm or 280 nm. Key instrument settings:

• Capillary length: 33 cm total with short-end (8.5 cm) or long-end (24.5 cm) detection
• Temperature: 20–35 °C
• Voltage: –20 to –30 kV

Key results and discussion

The study revealed several practical insights:

• Detection filter: Both 260 nm and 280 nm filters yielded six distinguishable peaks for the DNA ladder, with 260 nm providing superior signal-to-noise for poly(dA) sequences.
• Sieving matrix: All three PEG sources performed comparably; addition of 20% acetonitrile enhanced resolution for longer oligonucleotides.
• Capillary selection: PVA and µSIL WAX capillaries offered higher signal intensity than µSIL DNA, with relative standard deviations below 1% for migration times.
• Detection geometry: Short-end detection enabled rapid screening, while long-end detection produced higher resolution at the expense of analysis time.
• Temperature and voltage: Lowering temperature improved resolution but increased run time; higher voltage sharpened peaks and accelerated separations, balancing throughput and resolution.

Benefits and practical applications

This CGE approach on the Agilent 7100 system facilitates robust and reproducible sizing of oligonucleotides with single-base resolution. Laboratories can rapidly optimize separation conditions for diverse oligo mixtures, supporting applications in primer quality control, mRNA poly(A) tail analysis and therapeutic oligo characterization.

Future trends and applications

Emerging developments may include microchip-based gel electrophoresis for higher throughput, novel polymer matrices tailored to specific oligonucleotide chemistries, integration with mass spectrometry, and advanced multi-wavelength or laser-induced fluorescence detection to expand sensitivity and multiplexing capabilities.

Conclusion

The systematic evaluation demonstrated that through judicious selection of sieving gel composition, detection filter, capillary type, temperature and voltage, high-resolution separation of oligonucleotides can be achieved on a single CE platform. The Agilent 7100 CE system offers a flexible and reliable solution for routine oligo analysis.

References

1. Boden J, Meinka I, Meixner J. Oligonucleotide analysis with the Agilent Capillary Electrophoresis System. Agilent Technologies Application Note 5988-4303EN, 2001.