Sooner Scientific Catalog

Sooner Scientific Catalog

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Denaturing Gradient Gel Electrophoresis (DGGE) Systems

Denaturing Gradient Gel Electrophoresis System, 2 place. Cat. # SDGGE-2001

Description - Denaturing Gradient Gel Electrophoresis (DGGE) is a powerful genetic analysis technique that can be used for detecting single base changes and polymorphisms in genomic (1,2), cloned, and PCR amplified DNA (3,4). Two of the most valuable uses for DGGE in human genetics are in directly detecting single base changes that cause disease and in detecting polymorphisms with DNA probes for genetic-linkage analysis. Clinical applications of DGGE include a rapid and effective method for screening samples for genetic mutations. Also, DNA fragment melting points can be determined using Perpendicular DGGE (1).

The Sooner Scientific design has five different sized DGGE Systems which are reliable and easy-to-use. The DGGE-1001 is a smaller 2 gel system and features one dual gel cassette. The DGGE-2001 (shown above) has a larger buffer tank and features two single gel cassettes. The DGGE-2401 has the same buffer tank as the DGGE-2001 but features two dual gel cassettes, for a capacity of 4 gels per run. Also available is the DGGE-4001 which has a larger buffer tank and four single cassettes. Finally there is the DGGE-4801 which features the larger buffer tank and four dual cassettes enabling the researcher to run up to 8 gels simultaneously. Improvements to the technique include: a simplified method for casting perpendicular and vertical gels using Gel Cassette Sealer, single or dual gel cassettes have been redesigned eliminating the use of agarose plugs, the need for an external peristaltic pump for buffer cycling has been eliminated, bulky screw clamps have been replaced with polypropylene spring clamps, buffer tank dimensions have been changed to use bench space more efficiently, and a safety cover with an electrical interlock has been added for protection and to help maintain temperature and reduce evaporation.

The dual cassette system (as pictured above, Cat.# SDGGE-2001) consists of:

1 reinforced glass tank/lower reservoir with platinum electrodes with attached anode power lead
2 plexiglass gel cassettes/upper reservoir with platinum electrodes with attached cathode power leads
Safety Cover with Electrical Interlock
4 combs (2 each 1-well combs and 2 each 16-rectangular well combs.)
4 sets of spacers (2 with injection ports for perpendicular gel casting)
2 sets of glass plates
4 Gel Cassette Sealer gaskets
30 white spring clamps
Heater/Stirrer/Buffer Cycler
Gradient maker, 20mls per side
Buffer tank drain valve
Thermometer
SeaQuate acrylamide/bis 40% gel solution is shipped with each order. SQL 40191

Applications

Detecting single base DNA changes
Detecting Polymorphisms
DNA fragment melting points

Component Specifications of DGGE Systems

Buffer Tank Components: The reinforced glass buffer tank/lower reservoir can be ordered to accommodate 1 dual, 2 single, 2 dual, 4 single or 4 dual gel cassettes. The temperature control of the system has been optimized by adjusting the tank size to cassette displacement volume, and the dimensions have been designed so as to conserve bench space. A hinged safety cover with an electrical interlock protects against accidental contact with charged buffer, aids temperature control, and limits evaporation. The platinum anode is mounted in a electrode holder along the back vertical wall of the tank. Glass is the optimal tank material as it will not be distorted by the relatively high buffer temperature during electrophoresis (usually 60° C). Buffer tank drain valve and thermometer are included.
Power Supply: Sooner Scientific recommends using either Cat. # DOR-250 or DOR-250-II described here. The power supply is not included in the DGGE System price, and must be purchased separately.
Gradient Maker: The gradient maker is for gradient gel casting and comes with a 20ml volume per side. (Cat. #FL-40)
Single or Dual Gel Cassette(s)/Upper Reservoir: Single (see photo above right) or dual gel cassettes (see photo lower right) with upper reservoirs include a silicone gasket and platinum electrodes. The design of the cassettes allows both sides of the glass plates to be continually exposed to the circulating temperature controlled buffer. This feature minimizes temperature fluctuation within the gel/sample matrix and improves gel resolution. The base of the cassette fits into brackets in the bottom of the glass tank. White polypropylene spring clamps are provided for gel casting.
Heater/Stirrer/Buffer Cycler: The Sooner Scientific Heater/Stirrer/Buffer Cycler performs three functions:
- 1) Heating element and thermostat maintain excellent temperature stability (±0.2º) and minimize thermal stratification during DGGE.
- 2) Stirrer: The immersion-style impeller pump evenly distributes heated buffer.
- 3) Buffer Cycler: The system has been redesigned to eliminate the need of an external peristaltic pump to recycle buffer. Alternatively, each cassette has it's own upper to lower buffer cycling pathway with the buffer supplied via an internal bypass pump mounted on the Stirrer output.

Special Features

Extremely accurate and highly reliable technique for mutation detection (5, 6, 7).
This System lends itself well to the use of fluorescence based assays whose images can be captured on a fluorimeter directly through optional Borosilicate glass plate sets.

SDGGE-2001 kit components shown clockwise from lower left: glass plates, combs, spacers, 2 single gel cassettes, white clamps, gradient maker, and Gel Cassette Sealer. Dual cassettes are more efficient than single cassettes since they allow twice as many gels to be run in the same size buffer tank.

Accessories

Combs: A wide variety of combs are available for this system. Teflon® combs for gel thicknesses of 0.5mm and larger can be selected.
Glass Plate Sets: Notched glass plate sets are 17.7cm (w) x 22cm (h) (Cat. # MFO-17720-NR) and consist of one notched and one rectangular back plate. Borosilicate glass plates (Cat. # MFO-17720-B) are also available for direct scanning of fluroescent dyes using a fluorimeter. All glass plate sets are edge ground for safety and durability.
Perpendicular Comb and Spacer Sets: Specialized comb and spacer sets designed to simplify casting denaturing gradients perpendicular to the electrophoresis path are included with each DGGE System. Each set includes gel casting instructions and consists of: 1 standard spacer, 1 spacer with injection port, and 1 single well comb. These sets are available in thicknesses of 0.75mm and larger. This accessory eliminates the need to offset the side spacer during perpendicular gel casting.
Spacers: Heat stable Polycarbonate spacer sets can be ordered for gel thicknesses of 0.4mm, 0.5mm, 0.75mm, 1.0mm, 1.5mm, and 2.0mm.

Results

Ethidium Bromide stained DGGE gel of ß-Globin gene (from S.E. Asian population) after PCR of Fragment II. N= Normal allele, E= mutation on codon 26, 17= mutation on codon 17, Patient Control (IVS-1)= intervening sequence 1. Samples run on a 45%-75% gradient for 5 hours at 60°C at 200V constant current in TAE.

Personal Communication from Dr. Kim-Yen Ngo, U.C.S.D. Department of Medical Genetics.

References

S.G. Fischer and L.S. Lerman, (1983) PNAS 80:1579
R.M. Myers, T. Maniatis, L.S. Lerman, (1987) Methods in Enzymology, 155: 501-529
R.M. Myers, V.C. Sheffield, D.R. Cox (1988) "Genome Analysis: A Practical Approach," Ed. K Davies, IRL Press, Oxford. PP. 95-13
V.C. Sheffield, D.R. Cox, L.S. Lerman and R.M. Myers, (1989) PNAS, 86:232-236
Guldberg, P., Henriksen, K.F., and Guttler, F. (1993). "Molecular analysis of phenylketonia in Denmark: 99% of the mutations detected by denaturing gradient gel electrophoresis." Genomics 17:141-146
Moyret, C., Theillet, C., Puig, P.L., Moles, J-P., Thomas, G. and Hamelin, R. (1994). "Relative efficiency of denaturing gradient gel electrophoresis and single strand conformation polymorphism in the detection of mutations in exons 5 to 8 of the p53 gene." Oncogene 9:1739-1743
Sheffield, V.C., Beck, J.S., Kwitek, A.E., Sandstrom, D.W., and Stone, E.M. (1993). "The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions." Genomics 16: 325-332.

Ordering/Price Information - Please see page 6 or the index.

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SDGGE-2001 kit components shown clockwise from lower left: glass plates, combs, spacers, 2 single gel cassettes, white clamps, gradient maker, and Gel Cassette Sealer.	Dual cassettes are more efficient than single cassettes since they allow twice as many gels to be run in the same size buffer tank.