Cast of New Players: A Profile of New Precast Gels for Nucleic Acid Analysis

Date: June 7, 1999Precast Gel Table FMC Latitude™ Midigels Analyzing DNA on gels is not what it used to be. In the last two years, several new precast gels for nucleic acid analysis have been introduced to life scientists. Based on some novel and creative ideas, a few of these products have undoubtedly raised more than a few eyebrows. Others have probably generated comments like "It's about time." For example, it takes some adjustment to consider running a dry agarose gel--one that eliminates the need for slopping buffer around yet works just as well as the traditional submarine gel. Equally astounding is the concept of the reusable precast agarose gel. And how many researchers have longed for a precast sequencing gel? In this profile, LabConsumer rounds up the latest crop of precast gels designed for nucleic acid analysis. Made from agarose, acrylamide, and some novel matrices, these gels all succeed at streamlining and improving upon established techniques. Some clever and innovative ideas are to be found. The E-Gel™ Electrophoresis System from Invitrogen is the first bufferless precast agarose gel system available. Precast into a dry, disposable, UV-transparent cassette with electrodes, each gel consists of agarose, ethidium bromide, and a unique ion exchange matrix. This matrix has been designed to sustain the electric field and enhance the buffering capacity of the system without the need for submerging the gel in an aqueous buffer. Use of the E-Gel system is simple. After an E-Gel is removed from its storage pouch, it is snapped into the E-Gel base. The inexpensive E-Gel base then connects to a power supply and contains the gel during electrophoresis. Following a 2-minute prerun, the samples are then loaded and resolved in 30 minutes. Because ethidium bromide is already incorporated into the gel, the bands are stained evenly without the hazards associated with typical ethidium bromide staining procedures. Additionally, the gels are easily removed from the cassettes with the E-Gel Opener for band recovery or Southern blot processing. The elimination of aqueous buffers confers speed and convenience to the E-Gel system and makes for a device highly suitable for field use--a claim few systems make. E-Gels are sold in 1.2 percent, 2 percent, and 4 percent agarose concentrations, covering a DNA size range from 20 to 5,000 base pairs. A convenient starter pack, in addition to gel packages with up to 90 gels and other accessories, is available. On a similar note, Amersham Pharmacia Biotech's Flatbed Electrophoresis Systems make use of the first commercially available bufferless precast acrylamide gels. Featuring the GenePhor and Multiphor II platforms, the systems offer a broad selection of acrylamide gels in both bufferless (ExcelGel, GeneGel Excel) and semi-dry (CleanGel, GeneGel Clean 15/24) formats for high-resolution DNA analysis. The bufferless gels feature a ready-to-run system where no liquid buffer or acrylamide is ever handled for maximum convenience. The semi-dry gels feature a customized system where the precast gel matrix can be rehydrated using any buffer system desired. In addition, all of the flatbed gels are cast with a plastic backing for ease of handling and to eliminate the risk of tearing and distortion. Dedicated to the development of new materials and methodologies for the separation of biomolecules, Guest Elchrom Scientific AG of Switzerland has created three novel electrophoresis matrices, giving rise to its line of 55 different precast gels. Distributed in North America by AMRESCO, these gels fall into three types, each type designed for a specific application. Spreadex™ gels, for instance, resolve DNA bands within a narrow size range, Poly(NAT)™ gels provide high resolution across a broad size range, and Clearose™ BG gels offer quick runs and easy DNA fragment recovery. The fully synthetic Spreadex gels consist of a novel matrix of gel polymers arranged in a unique three-dimensional structure by free-radical polymerization. These gels offer superior resolving power by selectively retarding the migration of large DNA molecules so that the bands remain sharp but spread apart from each other. For example, Spreadex gels can resolve DNA bands differing by four base pairs on a 4 cm gel and by one base pair on an 8 cm gel. These gels are further characterized by an exclusion limit. That is, DNA fragments above a certain size will not migrate into a Spreadex gel, even after prolonged electrophoresis. Currently, these exclusion limits run from 300 to 1,200 base pairs. Bands excised from Spreadex gels may be used directly in PCR reactions because the matrix will not melt or inhibit the amplification reaction. Consisting of a monomer called N-acryloyl-Tris (NAT), a nontoxic derivative of Tris, the common buffer compound, Poly(NAT) gels are prepared by the polymerization of NAT in the presence of a cross-linker. The resulting gels provide 1 to 2 percent resolution of DNA fragments in the 100 to 2,000 base pair size range and are recommended for demanding applications such as the analysis of PCR products, heteroduplexes, RAPDs, and STRs. Overall, the three available concentrations of Poly(NAT) gels cover DNA fragments ranging from 10 to 20,000 base pairs. The optically clear Clearose BG gels are composed mainly of agarose cross-linked with butanediol diglycidylene. Tough and elastic, Clearose BG gels are the first reusable precast gels on the market. At the completion of a run, the DNA bands are driven out of the gel by reversing it in the electric field and running it for 10 minutes longer than the original sample resolution run. The gels are then reloaded with new samples and used again, up to about 10 times. Of the three types of Elchrom gels, Clearose BG gels are the best choice for applications requiring speed and reproducibility over resolution. Available with and without ethidium bromide, these gels handle DNA fragments spanning a size range of 50 to 10,000 base pairs, with optimal resolution falling between 200 and 2,000 base pairs. Finally, because Clearose BG does not melt at 100°C, DNA bands are easily recovered by diffusion at 70° to 75°C. These precast gels are all available in mini (9.2 x 6.2 x 0.3 cm) and wide mini (25.7 x 9.2 x 0.3 cm) formats with a variety of sample well configurations running up to 100 wells. While these gels run well in most common gel devices, they perform best in Elchrom's SEA 2000. The SEA 2000 is an advanced submarine electrophoresis device engineered with innovative features such as a built-in buffer recirculation pump and a heat exchange element. Not to be overlooked, AMRESCO has recently released a new line of precast polyacrylamide gels designed for vertical electrophoresis of proteins and nucleic acids in the common mini-vertical electrophoresis devices. Known as Multi-PAGE™ precast gels, the four gel types available resolve DNA fragments falling between 90 and 1,500 base pairs. 10% TBE Ready Gel, 9 well. Samples: lanes 1,5,7,9, AmpliSize Standard; lanes 2,4,8, 100 bp Molecular Ruler; lanes 3,6 Low Range Standard pBE322 digest with Avail and Avail/EcoR1. Image provided by Bio-Rad Laboratories. Introduced in 1993, FMC BioProducts' Reliant® gel system was the first line of precast agarose gels to gain acceptance in the marketplace and has since been credited with starting the precast gel rage. Today, Reliant gels consist of 1 percent SeaKem® Gold agarose and 4 percent NuSieve® 3:1 Plus agarose in either 1X TAE or 1X TBE buffer. The SeaKem Gold gels handle DNA fragments from 500 to greater than 10,000 base pairs, and the NuSieve gels accommodate fragments between eight and 1,000 base pairs. Sample formats include 1 x 8 wells and 2 x 12 wells in the "portrait" configuration and 1 x 20 wells arranged in the "landscape" configuration. Additionally, all gels are sold with 0.5 mg/ml ethidium bromide incorporated into the agarose; the eight-well gels are also sold without it. Reliant gels fit most common mini-chambers. For larger sample numbers, up to four Reliant gels can be placed side by side in larger horizontal gel boxes and run simultaneously. For the electrophoresis of RNA, FMC BioProducts manufactures the Reliant® RNA gel system. These precast gels are made with 1.25 percent SeaKem® Gold agarose in MOPS buffer without denaturants. Formulated to resolve RNA fragments from 250 to at least 10,000 bases, the gels are available with eight wells. And, like the Reliant gels above, multiple Reliant RNA gels may be run simultaneously. Lastly, because the gels are sealed in plastic trays, there is no need to treat gel devices to eliminate RNase contamination. Look for FMC to release a new product line of precast agarose gels called Latitude™ Precast Gels in the spring of 1999. Poured in a 10 x 15 cm size format, Latitude gels are prepared with ethidium bromide and will be available in a variety of buffer/well arrangements and agarose configurations. With over 170 separate precast gels, Embi Tec offers the largest number of gels on the market. Designed for Embi Tec's RunOne Electrophoresis System, which runs horizontal agarose and acrylamide gels, the RunOne™ Pre-Cast Gels fit other devices as well. Falling into three size categories--mini (5.9 x 5.2 cm), wide (5.9 x 10.7 cm), and wide-long (11.8 x 10.7 cm)--the gels consist of a choice of 16 sample well configurations in both agarose and acrylamide. All together, the gels tackle DNA fragments running from 20 to greater than 10,000 base pairs. In addition, some RunOne gels are shipped in UV-transparent trays for easy handling and viewing. Perhaps the most interesting feature of the RunOne gels is their unique sample well formatting. Thirteen of the 16 arrangements include separate reference wells for running DNA size markers, and many of the sample/reference well rows are arranged in tiers along the gel. For example, one 36-well format consists of four rows of eight sample wells plus one reference well per row. For added convenience, some of the sample well formats are compatible with multichannel pipettors. Embi Tec's 108-well gels are most notable here. One format consists of 12 rows of eight wells plus one marker well. These sample lanes correspond directly to the wells on a 96-well PCR sample plate so that the entire 96-well plate can be loaded quickly with a multichannel pipettor. The other 108-well format, six rows of 16 wells plus two reference wells, allows analysis of an entire 96-well plate while optimizing resolution. The Uni-Lane™ Gel SystemSM from Geno Technology Inc. offers researchers the luxury of using only the number of sample lanes required for a given experiment, thus eliminating wasted agarose from unused parts of poured gels. Best described as a single lane-by-lane format, Uni-Lane gels consist of trays of agarose sliced into 25 or 50 individual, single-sample-size gels and packed like bread slices in a bag. A researcher simply slides the number of gels needed (one per sample) off the storage tray one by one onto any horizontal electrophoresis gel box. After care is taken to align the gels in the electrophoresis device, they are loaded and run like any standard-sized agarose gels. Uni-Lane gels are composed of 1 percent, 2 percent, or 3 percent agarose in 1X TAE or 0.5X TBE buffers with ethidium bromide. They are sold in either 45 or 90 mm lengths along with the full line of Uni-Lane Gel System loading dyes, markers, and buffers. NOVEX, the industry leader in the precast gel market, has applied its expertise to the development of the QuickPoint™ Rapid Nucleic Acid Separation System. This system has been designed around QuickPoint Precast Gels. Composed of 6 percent acrylamide with a unique, neutral pH buffer formulation, QuickPoint gels resolve between 60 and 80 bases of DNA sequence and single-stranded products 600 bases or fewer in typical run times of 20 minutes. Overall, QuickPoint gels provide a quick and convenient electrophoresis method for nucleic acid analysis techniques that require such short read lengths. These techniques include site-directed mutagenesis, phage display, RNase protection assays, differential display, and other methods. QuickPoint gels are 10 x 12 cm in size with 16 preformed wells. The 0.25 mm thick gels are compatible with glycerol that would otherwise cause problems with many TBE-urea gel chemistries. On the whole, the QuickPoint separation system includes the necessary sample and running buffers and the QuickPoint Rapid DNA Sequencing Cell. Designed expressly for the QuickPoint gels, the QuickPoint cell runs one or two gels and is guaranteed never to leak. The CastAway™ Sequencing System manufactured by Stratagene consists of a flotilla of precast polyacrylamide gels and their support equipment designed for a battery of DNA analysis techniques. Resembling traditional, hand-poured sequencing gels with their 41.5 x 17.5 cm x 0.25 mm dimensions, the CastAway gels have a demonstrated usefulness with techniques such as DNA sequencing, RNase protection assays, and DNA fingerprinting. Other applications, such as the identification of differentially expressed gene products, are well documented in company literature. Stratagene's CastAway™ gel process Stratagene recently teamed up with LI-COR to develop the first precast gel for automated sequencing and genetic analysis. Designed for the LI-COR Model 4200 (IR2) Automated Sequencing System and manufactured with FMC's Long Ranger™ sequencing gel solution, these gels generate read lengths in excess of 500 bases. When all 32 sample wells are loaded with sequencing products, the gels produce over 16,000 bases of sequence data. The CastAway gels are available in 4.5 percent, 6 percent, 8 percent, and 12 percent acrylamide concentrations. Most gels have 32 preformed wells, although gels with a space for inserting a shark's tooth comb are available. Stratagene also sells a nondenaturing 6 percent gel for applications such as SSCP and differential display analysis where the complexity of the bands obtained from a denaturing gel may complicate the results. Invirtrogen's E-Gel™ and base The Genetic Systems division of Hitachi Software Engineering Company LTD sells the R3™ Precast Gel Electrophoresis System. This system uses a large-format 17.5 x 42 cm precast gel of 4.5 percent acrylamide gel designed to fit into the R3 Gel Box. Complete with 32 preformed wells spaced for compatibility with multichannel pipettors, the 0.25 mm-thick gels are designed for DNA sequencing, genotyping, STR, SSCP, and other applications. The procedures for using an R3 gel are spelled out in its name: Rip open the package, Run the gel, and Read the gel on Hitachi's FMBIO® II. The FMBIO II Fluorescent Imaging System exploits the latest fluorescence technology to increase the accuracy and efficiency of gel analysis. The laser incorporated into this device allows the excitation and detection of a broad spectrum of fluorescent dyes, allowing the FMBIO II to perform in a multicolored fluorescent environment. Numerous applications can be quantified, imaged, and analyzed by the FMBIO II, including SSCP gels, blots, and paternity testing and sequencing gels. The precast gel market has expanded in recent years with many companies offering complete lines of precast gel products.1 Among those are precast acrylamide gels prepared in TBE buffer for the analysis of nucleic acid fragments in the mini-vertical electrophoresis format. Not to be forgotten or overlooked, these precast gels remain valuable and useful in the life science laboratory. In fact, the gels and their resultant popularity have proven that the usefulness and convenience of precast gels are here to stay and that we can expect to see even more new products in the future. M. Brush, "Prepare to cast off: a profile of precast polyacrylamide gels," The Scientist, 12[15]: 18, July 20, 1998. The author can be contacted at Mdbrush@compuserve.com.

Cast of New Players: A Profile of New Precast Gels for Nucleic Acid Analysis

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