Colony Hybridization



DNA hybridization tests may be performed in many ways. An aliquot of a homogenized food(Golden rice) is spread-plated on an appropriate agar then incubated. After incubation, the colonial is being transferred into a solid support (usually paper filter that acts like a membrane) by pressing the support onto the agar surface. Next, the cells are lysed in situ by a combination of high pH and temperature (0.5 M NaOH and/or steam or microwave irradiation). This harsh environment denatures and affixes the DNA to the support. The solid support with the attached target DNA is incubated with a 32P- or enzyme-labelled probe. The failed labelled probe DNA will be removed by washing the probe-target complexes on the support at an appropriate temperature and salt concentration.

Great care must be taken to ensure that the washing temperature is correct. If the temperature of the washing solution is too high, all the hydrogen bonds between the probe and target may be broken. Thus, false-negative result is noted. On the other hand, if the washing temperature is too low, strands of DNA will not match up accurately, and non complementary strands may be formed. Thus,false-positive outcome is noted. If the temperature allows only accurately rejoined strands to remain together, the conditions are termed "high stringency." If the temperature is too low, so that mismatched strands exist, the stringency is low. For a review of hybridization using solid supports.(Food and Drug Adminstration 2009)


Autoradiography is used to detect Radioactive probe DNA that is bound to the target on the support. An X-ray film is placed over the support. Radioactive decays expose the film. This is because, when the films are developed, the black spots that appeared will be the cells that contain the same gene as the probe. Chromogenic substrate is added and an enzyme labelled probe is used. A colored spot will develop if probe-associated enzyme is present; Each spot represents a bacterial colony that has arisen from a single cell. The number of cells harboring the target gene in the original sample can be calculated by multiplying the number of spots by the dilution factor. (Food and Drug Adminstration 2009)

Food and Drug Administration (2009) BAM: Identification of Foodborne Bacterial Pathogens by Gene Probes. Retrieved on 30th June 2010 from http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/UCM072659


Youtube(2008)DNA Test Methods - Fluorescent In Situ Hybridisation . Retrieved on 1st july from http://www.youtube.com/watch?v=BBQWWi6cFXU&feature=player_embedded


Posted by: Alicia Chee