Denaturing Gradient Gel Electrophoresis and Temperature Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE)34,35 and temperature gradient gel electrophoresis (TGGE)36,37 are similar methods for separating DNA fragments with similar lengths but with different sequences according to their mobilities under a linear gradient of increasingly denaturing conditions. The gradient is created in DGGE with a mixture of urea and formamide, and in TGGE with a combination of water baths and a cooling plate under the gel. Both DGGE and TGGE take advantage of the markedly decreased mobility of partially melted dsDNA compared to either fully annealed dsDNA or ssDNA. Melting within a dsDNA fragment occurs within stretches of base pairs called melting domains. The point at which a domain begins to denature is referred to as the melting temperature (Tm), whether melting was induced by temperature or denaturing chemicals. In general, GC-rich sequences are more resistant to denaturation because of the three hydrogen bonds holding G and C together, as opposed to the two hydrogen bonds between A and T. During electrophoresis, once a dsDNA fragment reaches the point at which the melting domain with the lowest Tm begins to denature, mobility of the fragment through the gel nearly ceases. Fragments that melt early in the gel can therefore be separated from those that melt later. Complete denaturation of the dsDNA can be prevented by adding a GC-rich region to the 5' end of one of the primers (GC clamp), increasing the sensitivity for detection of sequence variants.

In DGGE and TGGE, the denaturing conditions and the time of electrophoresis should be optimized such that normal sequences migrate to an intermediate position in the gel by the end of electrophoresis. This allows sequence variants creating either a higher or lower Tm to be identified. The denaturing gradient may be perpendicular or parallel to the electric field. Perpendicular gradient gels covering a broad range of denaturing conditions are loaded with normal sequence in all lanes to find the optimal, narrower denaturing gradient (chemical or temperature) for later use in parallel gradient gels. Parallel gradients are then used to run samples but also to optimize the time of electrophoresis by loading the normal sequence to different lanes at different times. Double-gradient DGGE adds a sieving gradient, for example, 6% to 12% polyacrylamide, colinear with the denaturing gradient in the gel matrix, further improving band resolution.

Both DGGE and TGGE work best with DNA fragments less than 500 bp in length. When GC-clamped fragments are analyzed, the sensitivity of detecting a SNP is close to 99%. Following electrophoresis, specific bands can be isolated from the gel and sequenced. DNA fragments with a high GC content are not easily analyzed by DGGE, since all fragments are harder to melt.

Examples of Applications of DGGE or TGGE

1. APC gene mutation analysis in familial adenomatous polyposis38

2. CTFR gene mutation analysis in cystic fibrosis39

3. TCRG gene rearrangements in lymphoma40

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