A bioanalyser used in conjuction with a kit to size DNA fragments generated by restriction enzyme digestion can overcome limitations of slab gel electrophoresis. Odilo Mueller reports.
Slab gel electrophoresis (SGE) is currently the most widely used technique for restriction fragment analysis. While SGE is a relatively inexpensive and easy-to-use technique, the amount of accurate sizing information that can be derived without additional effort from slab gels is limited. Typically, the fragment length is estimated by the scientist through visual comparison to appropriate sizing ladders, which are run in separate lanes on the gel. Gel-scanning systems are available for these applications. They are used to scan gels after staining with an appropriate dye. However, these systems are expensive, require manual intervention, and the use of several individual hardware components.
The limitations of slab gel electrophoresis can be overcome with the Agilent 2100 bioanalyser, which is the first commercially available chip-based nucleic acid separation system. This system performs capillary electrophoresis on microfabricated channels and carries out detection as well as online data evaluation in an automated manner. The Agilent 2100 bioanalyser is connected to a PC for run control and automated data analysis. Several kits are available to analyse a variety of nucleic acid sample types. The DNA 12000 LabChip kit is best suited to the size determination of restriction digests with fragment sizes in the range of 100 to 12000 base pairs. Since restriction digests yield fragments with the same molarity, quantitative analysis is usually not needed.
Analysis of restriction fragment digests on the Agilent 2100 bioanalyser provides several important advantages compared to traditional slab gel electrophoresis. Since a short separation channel is employed and a high electrical field strength is applied, the speed of analysis is dramatically increased compared with slab gel electrophoresis. The speed of analysis results in an increased sample throughput. The instrument is equipped with a fluorescence detection system resulting in superior detection sensitivity. The prepackaged reagents and kits are used in conjunction with standardised protocols and result in more reproducible data. These kits also help to improve the overall reproducibility between different runs, chips, and instruments. Compared with data assessment with gel scanning systems, the amount of manual work is significantly reduced and even data analysis is performed in an automated manner. Sample and reagent consumption in the range of one to a few microlitres minimises exposure to hazardous materials and reduces the amount of waste material.
All chip-based separations were performed on the Agilent 2100 bioanalyser, which was controlled by dedicated software running on a PC. The Agilent 2100 bioanalyser software package includes data collection, presentation and interpretation functions. Data can be displayed as a gel-like image and/or as electropherogram(s) as shown inFig. 1. Additionally, sizing data is presented in tabular form and can be easily exported to various spreadsheet programs. A number of software tools are available for data manipulation and comparison. The Agilent 2100 bioanalyser contains high voltage power supplies, each of which is connected to a platinum electrode. These electrodes allow the instrument to perform multiple injections and other fluid manipulations from specific sample wells. The instrument uses fluorescence detection, monitoring fluorescence between 670 and 700nm.
Chip preparation
All chips were prepared according to the instructions provided with the DNA 12000 LabChip kit. Each kit includes 25 chips and the following reagents: sample buffer, gel matrix, dye concentrate, DNA markers, DNA sizing ladder, syringe, and spin filters. The gel-dye mix was prepared by mixing 400µl of the gel matrix with 20µl of the dye concentrate and the mixture was filtered through a spin filter. Next, 12µl of the markers were mixed with 60µl of the sample buffer. The separation chip was filled with the gel matrix/dye mixture and 5µl of the mixture of buffer/markers were added to each sample well. After adding 12 samples (1µl each) to the sample wells and the DNA sizing ladder (1µl) to the assigned ladder well, the chip was vortexed and run on the Agilent 2100 bioanalyser.
Adenovirus 2 DNA was purchased from Sigma-Aldrich Corp (St Louis, MO). The restriction enzymes, as well as the 1kbp ladder, were obtained from New England Biolabs Inc (Beverly, MA). Adenovirus 2 DNA was digested with Dra I or Bgl II using standard conditions, at a final DNA concentration of 50ng/µl. Following digestion, restriction enzymes were inactivated by the addition of EDTA to a final concentration of 10mM. Aliquots of 1µl were analysed on the Agilent 2100 bioanalyser using the DNA 12000 LabChip kit from Agilent Technologies GmbH.
The Agilent 2100 bioanalyser analyses 12 DNA samples in less than 30 minutes in a sequential manner and the results for each sample can be viewed after completion of the run. The DNA 12000 assay can be used to size double stranded DNA fragments ranging in size from 100 to 12000 base pairs, with a sizing accuracy better than 85 per cent. Run to run and chip to chip reproducibility is ensured by means of external standards (DNA sizing ladder) and internal standards (DNA markers). The assay is compatible with commonly used restriction digest buffers so that no desalting or other sample pre-treatment is necessary. Some restriction digests may require dilution of the sample if the total fragment concentration significantly exceeds 50ng/µl. While agarose gels often show good performance in certain size ranges, the DNA 12000 assay allows accurate sizing over a wide range of fragment lengths.
Chip versus gel analysis of 1kbp ladder and restriction enzyme digests of adenoviral DNA. Sizing accuracy was checked by running a DNA size standard and different restriction fragment digests on a one per cent agarose gel as well as on the Agilent 2100 bioanalyser. The samples contained various fragments ranging from 119 to 10000 base pairs in length. The individual fragments of each sample are listed in Table 1. For the gel analysis, a one per cent agarose gel stained with ethidium bromide was used, and the results are displayed in Fig. 2. In general, the fragments of all samples were separated, with only four co-migrating fragments (fragments 5088 and 5228 and fragments 1547 and 1549 in Adenovirus 2/Bgl II). However, to separate the longer DNA fragments, the runtime had to be extended which resulted in the shorter DNA fragments running off the gel and not being detected.
Assay action
The same three samples were also analysed using the DNA 12000 assay. No sample pre-treatment was necessary prior to loading the digested samples onto the chip (Fig. 3). While the chip-based separation showed similar resolution to the slab gel method (only one additional fragment was not separated), even the short fragments were detected in the sample. This improvement versus the slab gel method stems from the fact that all fragments pass by the detection window. The dynamic detection range of the detection system in the Agilent 2100 bioanalyser is sufficient to detect even minute amounts of sample (LOD _ 0.05ng/µl). For the three samples, the sizing accuracy was greater than 92 per cent. Typically, sizing accuracy is above 90 per cent. In the rare case where DNA fragments exhibit strong sequence-dependent migration, sizing accuracy remains greater than 85 per cent.
The wide range of DNA concentrations and DNA fragment sizes that can be analysed on a single chip makes the DNA 12000 assay a versatile and easy-to-use tool for the analysis of restriction fragment digests. Additionally, the automated online size analysis and tabular output for each sample is convenient and saves time compared with conventional gel technology.
Conclusion
The Agilent 2100 bioanalyser shows excellent performance for sizing of restriction fragments. The use of internal and external DNA markers allows analysis of multiple samples through a single separation channel with very high reproducibility and reliability. Data precision is comparable or superior to slab gel analysis, whereas analysis times are greatly reduced using the bioanalyser. Automation of both separation and data analysis makes this instrument versatile and easy to use. In addition to the analysis of restriction fragments with the DNA 12000 LabChip kit, the bioanalyser platform can be used for other nucleic acid analyses. Initial kits are available for sizing and quantitation of PCR fragments (DNA 7500 LabChip kit) and for quantitation and integrity/purity check of total RNA and mRNA samples (RNA 6000 LabChip kit).
Odilo Mueller is an application chemist for Agilent Technologies, Waldbronn, Germany. www.agilent.com
