Electric birefringence detection and analysis of DNA.

Abstract

Electric birefringence of DNA is studied as a means of detecting and sizing DNA fragments. Electric birefringence frequency dispersion (EBFD) of unseparated and unstained mixtures of restriction enzyme fragments in gels provides molecular weight information. Electric birefringence of DNA in electrophoresis gels provides real time, stainless densitometry at conventional electrophoresis conditions. Monodisperse DNA fragment rotational relaxation time constants have traditionally been measured by transient electric birefringence (TEB). The relaxation time constant is a measure of DNA mobility and provides the same molecular weight information as electrophoresis. Extraction of a single fragment relaxation time constant from TEB measurement of multi-fragment mixtures is difficult. EBFD measures individual rotational relaxation time constants of multiple fragments in gels. EBFD measurements of lambda phage Hind III fragments in agarose gel and phi X Hae III fragment in polyacrylamide gels are presented. EBFD measurement time for a five and half decade frequency sweep is just under five minutes. DNA fragments interact with the host gel when aligned in an electric field. EBFD is used to study the effect of DNA-gel interaction in monodisperse restriction fragments in the 4-23 kbp range. A model is presented which correctly predicts the effect of gel relaxation on multi-fragment EBFD measurements. EBFD of DNA in polymer solutions is presented as a possible rapid alternative to electrophoresis. A total analysis time of less than three minutes is possible for EBFD of a mixture of DNA fragments in polymer solutions. Visualization of DNA fragments in electrophoresis gels is usually accomplished by staining the DNA with a fluorescent dye or attaching a radio isotope label. Both techniques are time consuming and denature the DNA. On line, real time, stainless electric birefringence densitometry of DNA in electrophoresis gels is presented. Electric birefringence images of DNA fragments in whole gels are obtained with a CCD camera in three seconds at 10 V/cm. Detection limits are within an order of magnitude of ethidium bromide fluorescence densitometry. Accurate single fragment extractions using electric birefringence images are presented. The future of EBFD and electric birefringence densitometry in nucleic acid research is discussed.