Modifications for the Improvement of TGGE and DGGE

Abstract

Adding a 20–40 base-long stretch of guanosine and cytosine residues to the 5′ end of one of the PCR primers leads to a PCR fragment with one extremely thermostable end and shifts the irreversible transition during DGGE and TGGE to a higher temperature or higher denaturant concentration, respectively (9). The GC clamp remains base-paired, whereas the rest of the molecule becomes single-stranded. This state of partial denaturation results in the strong retardation of the nucleic acid’s mobility in the gel (cf. Fig. 8.4.1). The first, reversible transition is not usually influenced by this modification. However, segments which would normally denature in the irreversible transition may, instead, develop a second reversible transition when a GC clamp is present.