Verwendung der „Denaturierenden Gradienten-Gelelektrophorese“ (DGGE) zur Erfassung der mikrobiellen 16S-rDNA-Diversität in der Rhizosphäre

Abstract

Bacterial species can be separated according to the GC content of a highly variable region of the 16S ribosomal DNA using denaturing gradient gel electrophoresis (DGGE). This region is amplified with specific primers in a polymerase chain reaction (PCR) and amplification products are then loaded on the DGGE gel. The concentration of denaturants increases from the top to the bottom of the gel. While double-stranded DNA can migrate in the gel, gel denatured, i. e. partially opened DNA can no longer migrate and forms a band. Due to the presence of three hydrogen bonds between G and C compared to the two hydrogen bonds between A and T, GC-rich sequences are more stable. Thus, GC-rich sequences migrate further in the gel than GC-poor sequences. As bacterial species differ in the GC content of the amplified region, a characteristic band pattern is formed according to the species composition. After digitalisation, these patterns can be compared statistically. Our studies indicate that in barley not only root zones differ in the compostion of the bacterial population in the rhizosphere but also the iron status of the plant strongly influences the species composition.