Sample Preparation

Samples run on Capillary electrophoresis (CE) instruments are amplified as described in course: DNA Amplification, the Multiplexing Module before placing them into the autosampler tray.

Electrokinetic Injection

There are three methods used for sample introduction in CE systems:

• Hydrodynamic or pressure injection
• Siphoning
• Electrokinetic injection⁰⁵

All of these methods require the immersion of the capillary end into the sample. Electrokinetic injection is the only method used in forensic DNA analysis.

The DNA sample is loaded into the capillary separation matrix by electrokinetic injection. The process consists of the transfer of negatively charged ions via an electromotive force. As current flows from the cathode to the anode, the DNA sample is introduced at the cathode end of the capillary. Because only negatively-charged ions are transferred from the sample in the process, there is no significant loss of sample volume.

This type of transfer is directly influenced by the ionic strength of the sample.⁰⁶ The magnitude and duration of the voltage applied to the capillary during electrokinetic injection is directly proportional to the amount of DNA loaded into the capillary. Ideally, only the DNA present in the sample will contribute to the ionic strength. The presence of competing ions other than DNA rapidly degrades or prevents the injection process.

Electrokinetic injections are highly efficient in separating the DNA fragments due to the sample stacking in the capillary. Sample stacking in the capillary results when samples are injected from a solution that has a lower ionic strength than the buffer inside the capillary. When the electric field is applied during an electrokinetic injection, the resistance and field strength in the sample plug region increase because there are fewer ions to carry the current in the lower ionic strength samples. This causes the ions from the sample to migrate rapidly into the capillary. When these sample ions enter a region where the polymer solution and buffer are at higher ionic strength, they slow down and stack as a sharp band at the boundary between the sample plug and the electrophoresis buffer (Butler 1995).

Back Forward