ABSTRACT
Electric birefringence imaging (EBI) maps stresses in agarose electrophoresis gels. Both DNA and gel alignment can be monitored using this technique. Previously, we have reported birefringence images associated with resolved small (< 48 kbp) and partially resolved large 48 kb-(2 Mbp) DNA fragments oriented with relatively short electric field pulses. In this paper, differences between images acquired using short and long (DC) voltage pulses are presented. It is shown that stress signals distribute over long distances when exposed to prolonged electric fields. Resolution also affects band shapes. Refinements in instrumental design and evaluation of limiting noise sources of the technique are also described.
Subject(s)
Birefringence , DNA/isolation & purification , Electromagnetic Fields , Electrophoresis, Agar Gel , Electricity , Linear Models , Nephelometry and Turbidimetry , Temperature , Time FactorsABSTRACT
Image processing techniques which separate the DNA Kerr effect and induced electrokinetic distortion contributions to electric birefringence images of agarose nucleic acid electrophoresis gels are described. Under standard electrophoresis conditions, detection limits of 10 ng of DNA per well are obtained in hydroxyethylated agarose without signal averaging or 7.5 ng with averaging of four measurements. Maintaining constant gel temperature is shown to improve the quality of the images. Monochromatic light (589 nm) is shown to give a small increase in sensitivity compared to polychromatic (650 +/- 20 nm) light.
