To understand ITP, the name itself is a good place to start. Iso is the Latin term for same or equal, tacho is the Latin term for speed, and phorēsīs the Greek word for transmission. Bring that together and “isotachophoresis” is the separation of molecules moving at the same speed.
What is ITP?
Electrophoresis occurs when an electric field is applied to a solution containing ions. In the presence of the electric field, ions migrate through the solution in different directions at different speeds. ITP occurs when an electric field is applied to analyte of interest that is placed between two buffers, one containing ions with an electrophoretic mobility, or speed in the electric field, that is higher than the analyte and the other containing ions with a mobility slower than the analyte.
How is ITP used on the Ionic system?
To implement ITP on the Ionic system, we use a fluidic chip composed of 8 independent channels that are each connected to a series of wells that open to the surface of the chip. The figure below is a conceptual representation the separation process that occurs within each channel.
Prior to starting a run on the Ionic system, two primary buffers are loaded into separate wells. The first, shown in red above, contains a high concentration of “trailing” ions that have a slightly slower electrophoretic mobility than the nucleic acid, and the second, shown in blue above, contains a high concentration of “leading” ions that have an electrophoretic mobility faster than nucleic acid. In a priming step, the buffer in these wells moves into the fluidic channel filling it completely. Sample lysate is then loaded into another well of the chip that feeds into the same channel between the trailing and leading ion buffers.
At the start of a run, when electrical current is applied to opposite ends of the channel, ions begin to move through the solution. Positively charged molecules, such as metal ions, move toward the negative end of the channel. Negatively charged molecules including nucleic acids move toward the positive end of the channel. As this happens, the leading and trailing ions form a sharp electric field gradient, and nucleic acid moves toward this gradient. Nucleic acid gathers at the interface between the leading and trailing ions. This is commonly referred to as the LE/TE interface or ITP zone. Molecules or impurities with faster or slower electrophoretic mobilities are not bracketed by the leading and training ions and cannot enter this zone, effectively isolating the nucleic acid. As the nucleic acid continues towards the positive end of the channel it continues to concentrate within the ITP zone.
The tight band of nucleic acid eventually passes into a well where it can be collected. Using a sensor built into the instrument, the current is turned off once the nucleic acid enters this well. At that point, pure nucleic acid can be aspirated from the well using a standard laboratory pipette.
Since the separation occurs in a free solution, there is minimal shear force that can damage or fragment the nucleic acid. Furthermore, all nucleic acid present in the lysate is separated and collected regardless of fragment length or sequence. This results in an unbiased extraction and purification of nucleic acid with higher purity and yield than most conventional purification methods.
Where can I find more info?
For a deeper understanding of isotachophoresis, we recommend these publications: