Electric birefringence imaging of DNA in agarose electrophoresis gels.

Abstract

Electric birefringence imaging (EBI) provides sensitive, non-invasive detection of double-stranded DNA in agarose gels. Quasi-monochromatic, visible light is transmitted through an electrophoresis gel which is placed between plastic film polarizers. A slow-scan video camera equipped with a 12 bit A/D converter records the images. Signals in EBI are produced from intrinsic and induced birefringence arising from both DNA and gel. Under electrophoresis running conditions, hydrodynamically-induced gel distortion is shown to be the major source of birefringence for fragments smaller than 23 kbp. The birefringence generated approximates the DNA concentration gradient in the electric field direction. The stress-optic coefficient of 1% agarose gel is measured by mechanical compression and used to evaluate the magnitude of the induced stress on the gel during electrophoresis. Multi-linear regression analysis is used to quantitatively test the model for EBI signals. Birefringence attributed to localized electro-kinetic gel distortion and to intrinsic DNA birefringence is studied by fitting ethidium bromide fluorescence profiles to EBI results. Fluorescence polarization imaging is used to assess the influence of localized gel distortion on nucleic acid orientation across a fragment band. It is shown that DNA aligns parallel, on average, with an applied electric field independent of its location within a band. DNA orientation varies as the 1.2 power in electric field strength under the conditions tested. Both EBI sensitivity and quantitation are improved through image processing techniques which separate the DNA Kerr effect and induced electrokinetic distortion contributions. Under standard electrophoresis conditions, detection limits of 8 ng DNA per well are obtained in hydroxyethylated agarose without signal averaging. Maintaining constant gel temperature is shown to improve the quality of the images. Stress patterns in agarose gels during DC and field-inversion gel electrophoresis (FIGE) of nucleic acid fragments of varying sizes are mapped using EBI. Anomalies in DNA distribution are shown to result from spatial variations in hydrodynamic flow in the gel. In addition, on-line EBI monitoring during FIGE of megabase pair DNA size standards is demonstrated.