Gel Doc Data

Results

FIGURE 1. Application of GeneFLash Gel Documentation system for assays of topo I.

TopoGEN’s Topo I assay kit (1015-1) was used to measure relaxation of topo I. Increasing amounts of topo I (2005H-2)) were incubated with 250 ng pHOT1 supercoiled plasmid in duplicate 30 ul reactions for 15 min at 37o C. Reactions were terminated with stop buffer plus marker dye (SDS+ bromophenol blue). Panel A: Reaction products analyzed on a 1% agarose gel (TAE buffer, no ethidium bromide). Panel B: Reaction products analyzed on the same gel except 0.5 ug/ml ethidium bromide was included in the gel and TAE buffer. Gels were stained and destained with ethidium bromide after electrophoresis s at 2 v/cm until the dye reached the end of the gel. Each gel was then digitally documented using GeneFlash and the data are shown in Panel A and B. Band intensities were quantified using GeneFlash bundled software (GeneTools). Using GeneTools, the supercoiled band was assigned a value of 250 ng and other bands were measured against this value. Panel C shows a plot of the resulting values.

Discussion.

GeneFlash and Topo I Relaxation Assays.

Topo I engages duplex DNA in a cycle of cleavage and religation thereby converting a supercoiled circular DNA into a relaxed circular product. To measure the reaction and quantify topo I activity, TopoGEN’s topo I assay kit (1015-1) is used in conjunction with gel documentation (GeneFlash) and quantitation software (GeneTools). Since the assay measures accumulation of relaxed DNA species, one simply assigns a base value to the substrate (supercoiled DNA) in the GeneTools software (DNA concentrations are provided with TopoGEN kits). In Fig. 1 and 2, each reaction contained 250 ng of total plasmid DNA and this value is assigned to the supercoiled band in the gel using the software. For more rigorous analyses, one can deduct the small amount of nicked DNA that contaminates the supercoiled substrate. For example in this experiment, GeneTools determined that about 2-4% of the DNA was nicked (see Fig. 1A left most lane, upper band) which corresponds to roughly 5-10 ng of nicked DNA. The data in Fig. 1A,B show the titration of topo I activity and band intensity analysis from the GeneTools software in Fig. 1C. The precise amount of relaxed DNA is most easily deduced from the EB gel system (Fig. 1B) which collapses the gaussian distribution of topoisomers into a single band (labeled as ‘relaxed DNA’). GeneTools quantifies the signal levels of this band quickly and efficiently using 250 ng as the amount of reference DNA in the supercoiled band. We have additional data showing that analysis of the data in replicate experiments is reproducible with errors less than 5% in repeat experiments.

GeneFlash and Topo I Cleavage Analyses.

It is well established that topo I poisons such as camptothecin (CPT) and its congeners act by stabilizing the cleavage intermediate as described elsewhere on the TopoGEN web site (see Literature Links). The cleavages can be difficult to detect in that the intermediates (nicked or linear DNAs) are typically low yield end products; thus, one needs to optimize detection methods. The GeneFlash system is ideal for this purpose due to its sensitivity and ability to quantify band intensities. Experimentally there are several key steps to detecting and quantifying drug stabilized cleavages:

Use excess enzyme (since the reaction is stoichiometric) and ensure that the enzyme has good catalytic activity. Note that each cleavage complex ‘consumes’ at least one enzyme molecule in the covalent complex.
Use a DNA target that has low background DNA (for topo I this means as little nicked DNA as possible). TopoGEN DNA substrates are certified to work with our enzymes and to possess minimal levels of nicked DNA contaminants. These nicked DNA contaminants cannot be totally eliminated but can be minimized with good technique and proper monitoring and screening.
Terminate reactions with SDS while the enzyme is catalytically active and digest with proteinase K to remove the contaminating bound topo molecules. Failure to remove the denatured topo molecule will affect electrophoretic mobility of the nicked DNA forms.
Optimize detection and quantitation of the cleavage bands using Gel Documentation equipment such as GeneFlash and GeneTools software. One significant advantage of GeneFlash is the ability to alter UV filters and use significantly more sensitive dyes for staining DNA such as SYBR Green (4 times greater sensitivity or SYBR Gold, 20 times more sensitive than ethidium bromide). These new DNA and RNA dyes are available from www.molecularprobes.com

Application of GeneFlash and GeneTools for Topoisomerase I Drug Screening and Catalytic Assays

Christine Furbee and Mark T. Muller

FIGURE 2. Application of GeneFLash Gel Documentation system for topo I cleavages.

Topo I drug screening kit (1018-1) was used to quantify CPT induced topo I cleavages with purified human topo I (2005H-2). Reactions contained with 250 ng pHOT1 supercoiled plasmid in duplicate 30 ul reactions for 15 min at 37o C. Components in each reaction are indicated above the lanes. Reactions were terminated with stop buffer plus marker dye (SDS+ bromophenol blue) and digested with proteinase K as described in the kit. Panel A: Reaction products analyzed on a 1% agarose gel (TAE buffer, no ethidium bromide). Panel B: Reaction products analyzed on the same gel except 0.5 ug/ml ethidium bromide was included in the gel and TAE buffer. Gels were stained and destained with ethidium bromide after electrophoresis s at 2 v/cm until the dye reached the end of the gel. Each gel was then digitally documented using GeneFlash and the data are shown in Panel A and B. Band intensities for nicked, open circular DNA were quantified using GeneFlash bundled software (GeneTools). Using GeneTools, the supercoiled band was assigned a value of 250 ng and other bands were measured against this value. Panel C shows a plot of the resulting values.