DCode™ Universal Mutation Detection System

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Overview


Single-Stranded Confirmation Polymorphism (SSCP)

Consistent Results With SSCP

SSCP is a widely used mutation screening method because of its simplicity. Experimental conditions cannot be predicted for a particular sample, so it is important to optimize gel electrophoresis conditions, including run temperature, to ensure the highest sensitivity (Orita et al. 1989, Ravnik-Glavac et al. 1994). A temperature-controlled buffer bath makes the DCode system ideal for SSCP.

  • Electrophoresis cooling tank with ceramic cooling fingers connects to standard external laboratory recirculating chillers
  • Temperature control module with stirrer, heater, and buffer-recirculating pump maintains uniform temperatures between 5 and 25°C
  • Reproducible run temperatures for consistent results
  • In place of the cooling tank, the DCode system may be put in a coldroom set to any temperature above ambient
  • 16 x 16 cm or 16 x 20 cm gel sizes simplify nonisotopic detection with silver or fluorescent stains


Amplified mutant and wild-type alleles of exon 8 from the p53 gene.
Separation by SSCP run at a constant 30 W for 3.5 hr at 8°C in 1x TBE on an 8% acrylamide/bis/gel (37.5:1) with 3.5% glycerol. Lane 1, undenatured mutant allele;lane 2, mutant allele; lane 3, wild-type allele; lane 4, undenatured wild-type allele.

References

Orita M et al. (1989). Dectection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86, 2766-2770.

Ravnik-Glavac M et al. (1994). Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum Mol Genet 3, 801-807.

 

Constant Denaturing Gel Electrophoresis (CDGE)

CDGE for Rapid Screening

CDGE is another method for high-throughput screening. The optimal concentration of denaturant to use for a CDGE gel is determined from a perpendicular gradient gel. Once the optimal denaturant concentration for a particular mutation is determined, multiple samples can be screened on a constant denaturant gel (Borresen et al. 1991). Thus, with CDGE, no chemical gradient is required and rapid high-throughput screening is simple.

  • DCode system for CDGE simplifies optimization of denaturant concentration
  • Optimized electrophoresis reagents and controls ensure quality results for high-throughput screening
  • Convenient 16 x 16 cm and 16 x 10 cm formats and simple tape-free gel casting facilitate rapid screening


Amplified alleles from the β-globin gene separated by CDGE on an 8% acrylamide gel in 45% denaturant at 56°C.
Lane 1, a compound mutant sample IVS1-1 + IVS1-6; lane 2, mutant sample IVS1-1; lane 3, mutant sample IVS1-6; lane 4, mutant sample IVS1-110; lane 5, wild-type DNA.

Reference

Borresen AL et al. (1991). Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc Natl Acad Sci USA. 88, 8405-8409.

 


Temporal Temperature Gradient Eletrophoresis (TTGE)

The Power of DGGE Without Gradient Gels

TTGE exploits the principles of DGGE without using chemical denaturing gradient gels, making it simpler, faster, and easily reproducible (Yoshino et al. 1991). Amplified DNA is loaded onto a polyacrylamide gel containing urea. During electrophoresis, the temperature is increased gradually and uniformly. The result is a linear temperature gradient over the course of the electrophoresis run. The denaturing environment is formed by a constant concentration of urea in the gel, combined with the temporal temperature gradient. The DCode system reduces TTGE to simple, reproducible practice.

  • Temperature controller and heater allow reproducible temperature ramps as low as 0.1°C per hour
  • Elimination of gradient gels makes setup and gel casting easy
  • WinMelt™ software helps determine optimal temperature ranges
  • Control reagents and application notes with proven protocols allow you to start experiments immediately

Amplified mutant and wild-type alleles of exon 7 from cystic fibrosis gene. Separation by TTGE run at 130 V for 5 hr in 1.25x TAE buffer on a 6 M urea/6% acrylamide/bis gel (37.5:1) using a temperature range of 50–60°C and ramp rate of 2°C/hr. Lane 1, mutant allele (1154 insTC); lane 2, mutant allele (G330X); lane 3, mutant allele (DF311); lane 4, mutant allele (R334W); lane 5, wild-type allele (samples courtesy of L Silverman, Division of Molecular Pathology, University of North Carolina School of Medicine).

Reference

Yoshino K et al. (1991). Temperature sweep gel electrophoresis: a simple method to detect point mutations. Nucleic Acids Res 19, 3153.

Denaturing Gradient Gel Electrophoresis (DGGE)

DGGE to Hunt for Unknown Mutations

DGGE is based on the principle that increasing denaturant concentration will melt double-stranded DNA in distinct domains. When the melting temperature (Tm) of the lowest domain is reached, the DNA will partially melt, creating branched molecules with reduced mobility in a polyacrylamide gel (Myers et al. 1987). The denaturing environment is created by a uniform run temperature between 50 and 65°C and a linear denaturant gradient formed with urea and formamide. The gradient may be formed perpendicular or parallel to the direction of electrophoresis. DGGE is one of the most sensitive mutation detection methods, providing efficiency up to 99% (Grompe 1993). The DCode system optimizes DGGE in the following ways:

  • Gradient gel casting is simple with the patented cam-operated Model 475 gradient former
  • WinMelt™ software streamlines GC clamp and primer placement
  • Temperature control module provides consistent run temperatures between 45 and 70°C
  • Run up to two 16 x 16 cm gels or four 7.5 x 10 cm gels

An example of a perpendicular denaturing gradient gel in which the denaturing gradient is perpendicular to the electrophoresis direction. This example shows a single melting domain. At low denaturant concentration (left) the DNA fragment remains double stranded, but as the concentration increases (moving right) the DNA fragment begins to melt, creating a branched molecule. At very high concentrations, the DNA fragment can completely melt, creating two single strands.


A, perpendicular denaturing gradient gel in which the denaturing gradient is perpendicular to the electrophoresis direction. Mutant and wild-type alleles of exon 6 from the p53 gene amplified from primary breast carcinomas and separated by perpendicular DGGE (0–70% denaturant), run at 80 V for 2 hr at 56°C (data courtesy of AL Borresen, Radium Hospital, Oslo, Norway). B, parallel denaturing gradient gel in which gradient is parallel to the electrophoresis direction. Mutant and wild-type alleles of exon 8 from the p53 gene, electrophoresed in an 8% acrylamide:bis (37.5:1) gel with a parallel gradient of 40–65% denaturant. Gel was run at 150 volts for 2.5 hours at 60°C in 1x TAE buffer. Lane 1, mutant fragment; lane 2, wild-type fragment; lane 3, mutant and wild-type fragments.

References

Grompe M (1993). The rapid detection of unknown mutations in nucleic acids. Nat Genet 5, 111-117.

Myers RM et al. (1987). Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol 155, 501-527.

DCode Applications

Screening Mutations Causing Cancer

Identification of mutations within specific genes has become an important strategy in determining the diagnosis and prognosis for many diseases, including cancer. Several techniques, including DGGE, CDGE, TTGE, and SSCP, have been developed to examine heterogeneous tissue samples for specific mutations. These techniques are useful for rapid screening of heterogeneous tissues, and each technique has its individual strength.

Any of the screening techniques listed above will identify known polymorphisms. This reduces the number of bands that must be fully sequenced, thus lowering the cost of analysis and increasing the overall efficiency of genetic screening. Here are examples of screening mutations such as p53, K-ras, and mutations in the Fas antigen of lymphoma tumors using the DCode system.


Parallel DGGE analysis of the human p53 gene using the DCode system. Lane 1, wild-type DNA of exon 5; lane 2, sample (breast cancer) DNA of exon 5; lane 3, wild-type DNA of exon 6; lane 4, sample DNA of exon 6; lane 5, wild-type DNA of exon 7; lane 6, sample DNA of exon 7; lane 7, wild-type DNA of exon 8; lane 8, sample DNA of exon 8. The arrow in lane 2 shows a mutation in exon 5 (see bulletin 2415).



Parallel DGGE analysis of the Fas III domain in lymphoma patients. Total RNA was isolated from lymphoma tumor tissues, reverse transcribed, and the Fas III domain amplified with 35 cycles of PCR. Products were analyzed on a 6% acrylamide DGGE gel. Lane 1, CEM (polymorphic); lane 2, 8226 (wild-type); lanes 3–12, lymphoma patient specimens (see bulletin 2295).



35% CDGE gel. Lanes 1, 2, 19, and 23, contamination control (aspirated buffer); lanes 3–17, mutant samples; lanes 20–22, wild-type control. Arrow: Heteroduplex band caused by mutated K-ras (see bulletin 2330).


Monitoring Microbial Diversity

Assessing bacterial genetic diversity in the natural environment is challenging because of difficulties in culturing native bacteria, and the many species encountered. Nucleic acid sequence comparison is the most fundamental way to classify microorganisms; however, the limitation is that DNA sequencing is expensive and laborious.

A new approach in microbial genetics is based on the analysis of bacterial genetic information without cultivation. This culture-independent approach has greatly enhanced the ability to assess bacterial genetic diversity in natural ecosystems and in mouth, intestinal, or other microflora.

The ubiquitous prokaryotic 16S ribosomal RNA gene has been the most widely targeted molecule for these studies because it has highly conserved nucleotide sequences. Variable regions of the 16S rRNA gene are amplified by PCR using universal primers. The DNA sequence in this variable region differs enough among organisms that it can be accurately used to identify a species (Avaniss-Aghajani et al. 1996). The mixture of amplified variable regions can be separated by DGGE, CDGE, or TTGE, and the resulting diversity patterns analyzed and compared. The major advantage of these techniques is that they allow direct determination of bacterial genetic diversity, making them superior to cloning and subsequent sequencing.





TTGE gel of Nitrosospira 16S rDNA PCR products amplified from monthly freshwater samples. DNA bands in positions A to F were excised and sequenced for the determination of phylogenetic relatedness (see bulletin 2427).








DGGE analysis of multiple competitive PCR products from mixed genomic DNA spiked with known quantities of standard after 35 cycles of amplification. Estimation of cell numbers from the 105 spike generated values in good agreement with that expected by microscopic counting prior to cell lysis (see bulletin 2525).




DGGE patterns from surface water demonstrating temporal changes in microbial populations. Surface water samples (the river Ruhr) from March–August (lanes 1–6, respectively); see bulletin 2366.


Reference

Avaniss-Aghajani E et al. (1996). Molecular technique for rapid identification of mycobacteria. J Clin Microbiol. 34, 98-102.

DCode Systems

The search for unknown mutations in genomic DNA is important for studying the genetic basis of diseases and disorders, including cancer. Additionally, examining DNA polymorphisms is useful for ecological and evolutionary studies of terrestrial, marine, and microbial organisms, with applications ranging from species identification to delineation of population structure to monitoring genetic diversity.

The DCode universal mutation detection system can scan for single-base changes by any of the following electrophoretic techniques:

  • Single-stranded conformation polymorphism (SSCP)
  • Denaturing gradient gel electrophoresis (DGGE)
  • Constant denaturing gel electrophoresis (CDGE)
  • Temporal temperature gradient gel electrophoresis (TTGE)

Flexible and powerful, the DCode system is the one electrophoresis system that can perform any combination of these techniques. At the center of the system is the temperature control module (which includes a microprocessor-controlled heater, a buffer-recirculating pump, and a stirrer). For techniques requiring accurate temperature control, such as SSCP and TTGE, the gels are immersed in the buffer, and temperatures are regulated between 5° and 70°C. Any run temperature below ambient can be achieved with the cooling tank used in conjunction with an external laboratory chiller. The DCode system can run 64 samples in as little as 2 hours — a major consideration when you're screening DNA for sequence variations.

You can configure your DCode system to grow as your needs grow. Each system includes a vertical electrophoresis cell and choice of adaptor kits for SSCP, DGGE, and CDGE, as well as for TTGE, a technique codeveloped by Bio-Rad. TTGE has all the benefits of DGGE and CDGE without chemical denaturant gradients.

This flexibility lets you quickly add new research tools without investing in new instrumentation. Systems configured for the primary application can be expanded to perform any combination of methods by supplementing the core system with technique-specific adaptor kits listed on the accessories tab. Product options include the complete DCode system and individual DCode systems for DDGE, CDGE, TTGE, and SSCP.

WinMelt™ Software

Mutations are most reliably detected when the sequence difference occurs in the lowest melting-temperature domain of the DNA of interest. In addition, optimal resolution is attained only when the molecules do not completely denature. The addition of a GC clamp onto one end of the DNA via PCR ensures that the region screened is in a lower melting-temperature domain, and that the DNA will remain partially double stranded. This enhances both detection and resolution. Windows-based WinMelt software predicts the melting profile of any DNA sequence up to 3,200 bases. Placement of primers and GC clamps can be optimized by analysis of their effect on the DNA melting profile.

Operation of WinMelt software is simple. The DNA sequence is imported from a text file and the melting profile is computed. The data appear onscreen and can be graphed according to user preference. The sequences and melt data can then be exported for use in other programs. WinMelt software is recommended for all DGGE, CDGE, and TTGE applications.

Electrophoresis and Control Reagents

Bio-Rad electrophoresis reagent kits are customized for each application to ensure the highest-quality buffers and acrylamide. Our control reagents for DGGE, CDGE, TTGE, and SSCP help you quickly master any new techniques.

Applications

Technical notes with proven DCode system run conditions are available. To obtain all DCode system application notes, request literature package 1720J. To obtain DCode application notes on microbial diversity studies, request literature package 1720K.

Training Guide

This self-training guide is an interactive CD-ROM that helps you learn about the techniques used to screen mutations using the DCode system. It includes the following:

  • Principles of DGGE, CDGE, TTGE, and SSCP
  • Videos on setting up and using the DCode system
  • WinMelt software tutorial program, DCode application notes, instruction manual, and other literature

To obtain your self-training guide, request catalog #170-9241. The CD-ROM is PC-compatible only.

Note to D GENE™ Users

All of the DCode adaptor kits are compatible with the D GENE apparatus, with the exception of the SSCP adaptor kit. To run gels below room temperature, place the D GENE system in a coldroom or refrigerator equipped with electrical outlets, and use the temperature controller to set the desired run temperature (see bulletin 1935). The basic SSCP adaptor kit is compatible with the D GENE system, but requires use of a coldroom for electrophoresis below room temperature.

DCode Universal Mutation Detection System  
Components  
Tank, core, and clamps Tank: molded polycarbonate; core: molded polysulfone; clamps: molded glass-filled polycarbonate
Gradient former Cast acrylic and aluminum
Lid Polycarbonate
Electrodes 0.010" diameter platinum
Electrical leads Flexible, straight
Glass plates 20 x 20 cm (20 cm format inner plate)
Gel sizes 16 x 20 cm (maximum two per run)
Spacers available 0.75, 1.0, 1.5 mm
Combs 16-, 20-, 25-, and 32- (1 mm only) well combs, and 1- well comb (prep comb for perpendicular gradient gels)
Casting stand Casts two 16 x 16 cm, two 16 x 10 cm, two 16 x 20 cm, or four 7.5 x 10 cm gels per setup
Heater and control Temperature control (PID type) ±0.5°C variation within gel area, ±0.5°C actual in the range of 45–70°C
Maximum set temperature 70.5°C
DC voltage/power limit 500 V DC voltage limit; 50 W power limit
Size and Weight   
Dimensions (W x D x H) Lid and tank assembly: 39 x 20 x 42 cm
Weight 16 kg (35.3 lb)
Power Requirements  
AC power requirement AC power input: 120 VAC 47–63 Hz, 5 A slow-blow fuse
(catalog #170-9080/9083/9086/9089/9092/9095/9098/9102)
DC power requirement External DC voltage power supply; power supply must be ground isolated in such a way that DC voltage output floats with respect to ground
Maximum voltage limit 500 V DC
Maximum power limit 50 W
Environmental Requirements  
Storage environment 0–70°C, humidity 0–95% (noncondensing)
Operating environment 0–35°C, humidity 0–95%
Regulatory Meets requirements of IEC 1010-1 and FCC, Class A
Model 475 Gradient Delivery System  
Size and weight 22 x 21 cm (W x H); 2.28 kg (5 lb)
Capacity 50 ml total (25 ml/syringe)
WinMelt™ Software  
System requirements 486 MHz or faster CPU, CD-ROM drive
DCode System for DGGE

170-9080
120 V, universal mutation detection system, for 16 cm gels with one 1 mm prep well, includes comb gasket, 2 sets of clamps, Model 475 gradient former with all parts required to cast gradient gels

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DCode System for DGGE

170-9083
120 V, universal mutation detection system, for 10 cm gels with two 1 mm prep wells, includes comb gasket, 2 sets of clamps, Model 475 gradient former with all parts required to cast gradient gels

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DCode System for CDGE

170-9086
120 V, universal mutation detection system, for 16 cm gels with twenty 1 mm wells, includes comb gasket, 2 sets of clamps, Model 475 gradient former with all parts required to cast gradient gels

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DCode System for TTGE

170-9089
120 V, universal mutation detection system, for 16 cm gels with twenty 1 mm wells, includes comb gasket, 2 sets of clamps, control reagents

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DCode System for SSCP

170-9092
120 V, universal mutation detection system, for 20 cm gels with twenty 0.75 mm wells, includes cooling tank adaptor for use with external cooling bath, control reagents

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Complete DCode System

170-9105
120 V, PC-compatible, complete universal mutation detection system, includes accessories, WinMelt software, control reagents for DGGE, CDGE, TTGE, and SSCP

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DCODE TEMP.CNTRL.MOD,100V

170-9078
DCODE TEMP.CNTRL.MOD,100V

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Number Description Options
4006138 Instruction Manual, WinMelt and MacMelt Software, Rev A Click to download
4100103 Instruction Manual, DCode Control Reagent Kit for SSCP Click to download
M1709080 Instruction Manual, DCode Universal Mutation Detection System, Rev D Click to download
5479 DCode System CD: Literature Library, Rev B Click to download
2101 DCode System and Accessories, Rev C Click to download
2100 DCode Universal Mutation Detection Systems, Reagents, and Accessories Ordering Information, Rev E Click to download
2069 DCode Universal Mutation Detection System Brochure, Rev C Click to download [ Add to Cart (Free) ]