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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 (T_m) 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, The rapid detection of unknown mutations in nucleic acids, Nat Genet 5, 111–117 (1993)

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