Using a Nonradioactively Labeled nifKDH Gene Probe to Locate Complementary Sequences of Rhizobial DNA Immobilized on Membranes

Abstract

In the Southern blotting procedure, the DNA separated on the agarose gel is denatured into single strands prior to transfer and immobilization on special nitrocellulose (NC) filters or nylon membranes. Because of the single-stranded nature of the immobilized DNA on the membrane, sequences complementary to the gene probe will lead to DNA-DNA hybridization between the membrane immobilized DNA and the denatured (single stranded) gene probe. The sites of hybridization on the membranes can be visualized using different procedures for the detection, depending on whether a radioactive or nonradioactive label was used on the probe. Signals from radioactively labeled probes are captured by exposing the NC filter or nylon membrane to X-ray films (autoradiography). Biotin-labeled probes are usually detected by the enzyme-linked immunoassay using an enzyme conjugate streptavidin-alkaline phosphatase (SA-AP). The streptavidin (a biotin-binding protein) part of the conjugate binds to the biotin. Adding a chromogenic substrate initiates a color reaction because cleavage by the enzyme produces a colored product. Colored bands become visible on the NC filters or nylon membranes at the sites of DNA-DNA hybridization. In this experiment, Southern blots of genomic or plasmid DNA on NC filters or nylon membranes are probed with a biotinylated nifKDH gene sequence and a color reaction indicates hybridization sites. A commercial kit for the nonradioactive labeling and hybrid DNA detection is used.