Basic Principles

The Bioanalyzer seperates nucleic acids (DNA and RNA), and proteins based on size, using the principles of electrophoresis.

Separation of nucleic acids using the Bioanalyzer is much like conventional agarose electrophoresis - the rate of migration of nucleic acids is based on the size of the molecule. Smaller size nucleic acids will migrate faster through a matrix in the presence of an electric field, relative to a larger size nucleic acid.

The negatively charged phosphates in DNA/RNA backbones result in nucleic acids having an overall negative charge. The cathode (negatively charged electrode) repels, and the anode (positively charged electrode) attracts nucleic acids.

Figure 1: The image above is a graphic representation of the electrophoresis. Smaller (shorter fragments) of DNA/RNA will migrate faster than larger (longer fragments) of DNA/RNA.

The main distinction between the Bioanalyzer and conventional agarose gel electrophoresis is the gel martrix that the nucleic acids migrate through, and the detection system which detects resolving nucleic acids. The gel matrix is contained in microcapillary chanels, which allows for the extremely sensitive quantification and sizing of nucleic acids. The microcapillary chanels are precisely manufactured into a glass slide, and placed into a Bioanalyzer chip.

The gel-dye matrix furthermore contains a proprietary intercalating agent that produces fluorescence when intercalated with DNA/RNA and excited by the excitation system. Relative fluorescence is measured, and the size of the nucleic acids are later assigned to fluorescence intensity by means of a standard ladder. The ladder is always run for every assay, just like normal agarose gel electrophoresis.

Figure 2: The microcapillary chanels in which the gel-dye matrix is loaded, and the samples resolved

Number of samples per run:

• DNA 1000, 7500,12 000, and RNA 6000 Nano : 12 samples/chip
• DNA High Sensitivity, Small RNA, and RNA 6000 Pico, : 11 samples/chip

Sizing of Proteins

Proteins can also be resolved using the 2100 Bioanalyzer.

The same principles of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are used.

Proteins samples are denatured and disulphide bonds reduced , eliminating any secondary and tertiary protein structures. The proteins are furthermore treated to have an overall negative charge, such that they might migrate in the presence of an electric field. Being repelled by the cathode and attracted by the anode.

Once again, the matrix and detection system of the 2100 Bioanalyzer allows for an extremely sensitive sizing and quantification method of resolved proteins.

The Bioanalyzer user guide can be found from the link below:

https://www.agilent.com/cs/library/usermanuals/public/2100_Bioanalyzer_Expert_USR.pdf