Interactive control of pulsed field gel electrophoresis via real time monitoring

Abstract

The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge-coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis is possible throughout the run via this detection scheme. The use of absorption removes the need for staining or radioisotope labelling, thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without destaining. The optimization of buffer concentration, electric field strength, temperature, agarose concentration, as well as pulse duration can considerably reduce total run time. For example, DNA from 2 to 850 kb can be separated in 3 h on a 7-cm gel with interactive control of the pulse time, which is 10 times faster than using a constant field program.

A CCD-based system for the detection of DNA in electrophoresis gels by UV absorption

“…This implies that DNA can be detected by UV absorption. This technique, known as UV shadowing, has previously been used for detection of nucleic acids in gels; however, experiments have resulted in detection limits of 2-3 µg (Hassur and Whitlock 1974), 0.13 µg (Clarke et al 1982) and 2 ng (McGregor and Yeung 1992) which are no better than the limits when using EtBr. Figure 1 shows the absorption spectrum of double stranded (ds) DNA.…”

Expert system for data acquisition to achieve a constant signal-to-noise ratio: application to imaging of DNA sequencing gels

“…An exception is in pulsed-field gel electrophoresis. 1 In multiplexed spectroscopy such as Fourier transform variants, all data points must be acquired before a valid transform is allowed. The distribution of S/N in the resulting spectrum does not follow a simple dependence on the S/N of the individual points in the interferogram.…”