Dot-Blot Hybridization Technique

Abstract

The dot-blot hybridization is a nucleic acid hybridization technique where complementary single-stranded sequences of the probe (either RNA or DNA) hybridizes with single-stranded sequences of the test samples (either RNA or DNA) under suitable conditions of temperature and salt concentration. It is based on the homology between two strands of nucleic acid (DNA:DNA, DNA:RNA or RNA:RNA). In this assay, a probe which is a single-stranded nucleic acid (either DNA or RNA) is ‘labelled’ with a reporter molecule. The labelled probe is then allowed to form hybrid with nucleic acid isolated from the test plant. The double-stranded hybrid molecule is then detected using appropriate method depending on the reporter molecule used. The process involves isolation of nucleic acid from test plant; it’s spotting on a membrane, pre-hybridization, hybridization using labelled probe and their detection. The DNA probes may be labelled using different methods, namely nick translation, random primed labelling or by polymerase chain reaction. The RNA probes are labelled by in vitro transcription. The labels can be either radioactive or non-radioactive. If radioactive probes are used, then the detection is done through autoradiography. If non-radioactive labels are used, then suitable detection methods through either chromogenic or chemiluminescence is used.