Bio-Rad Experion DNA Analysis Kits - Manual (Page 50)

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Appendix A: How the Experion ™ System Works
The Experion automated electrophoresis system performs electrophoresis of samples within a
microfluidic chip. Within each chip, a series of microchannels connects the sample wells to a separation
channel and buffer wells. A set of electrodes in the electrophoresis station applies a voltage across
the microchannels, causing charged molecules in the samples to migrate into and through the
separation channel. Samples are run sequentially, with a sufficient lag between them to prevent cross-
contamination. For separation, the microchannels are filled with a proprietary gel-stain solution (GS) that
acts as a sieving matrix; therefore, the sample DNA fragments migrate through the separation channel
at a rate based on their size and charge. Finally, the fragments interact with a fluorescent dye during
separation and are detected as they pass a laser and photodiode detector (laser-induced fluorescence).
DNA analysis is accomplished with the Experion DNA 1K and 12K analysis kits and involves the
following steps:
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Preparing the chip (priming and loading) — Priming fills the microchannels of the
microfluidic chip with GS, which contains both the sieving matrix and fluorescent dye.
DNA samples and loading buffer are then added
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Running the chip — The chip is inserted into the electrophoresis station, and as the
instrument lid is closed electrodes come into contact with the solution in the wells. Voltage
is applied to the sample wells of the chip, causing the charged DNA fragments to migrate
into the separation channel. In the separation channel, the different fragments separate
as they move at different rates through the gel matrix, depending on their size. During
separation, the fluorescent dye intercalates between the bases of the fragments
n
Detecting the fragments — As the fragments migrate toward the end of the separation
channel, a laser excites the dye, causing it to fluoresce if it is bound to the DNA. A
photodiode detects the fluorescence, and Experion software plots the fluorescence
intensity vs. time to produce an electropherogram and a virtual gel image
n
Analyzing the data — Following separation, Experion software subtracts background noise,
removes spikes, identifies and integrates peaks, and assigns their sizes and concentrations.
Following analysis, parameters may be changed and the data reanalyzed
How Experion Software Analyzes DNA Fragments
Data Presentation
As the photodiode detects fluorescent signals from the dye-DNA complexes, Experion software
converts the signal into an electropherogram (a plot of fluorescence vs. time). Experion
electropherograms generally have the following features (Figure A.1):
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Sample peak(s) — signal(s) generated by the sample fragment(s)
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Upper and lower markers — signals generated by the upper and lower markers, which are
included in the DNA loading buffer to normalize the separation of fragments across all wells
in a chip. The upper marker is also used as an internal standard for relative quantitation
(see below). For each lane, comparison of the corrected area of this peak with the
corrected area of every detected peak allows estimation of relative concentration
Once separation occurs, the data are converted into a densitometric, gel-like image, or virtual gel
(Figure A.2). Each lane in the virtual gel corresponds to a different sample. The sample peaks and upper
and lower markers seen in the electropherogram also appear in the virtual gel.
Experion Automated Electrophoresis System