The DNA of one organism is labelled, then mixed with the unlabelled DNA to be compared against. The mixture is incubated to allow DNA strands to dissociate and then cooled to form renewed hybrid double-stranded DNA. Hybridized sequences with a high degree of similarity will bind more firmly, and require more energy to separate them: i.e. they separate when heated at a higher temperature than dissimilar sequences, a process known as "DNA melting". To assess the melting profile of the hybridized DNA, the double-stranded DNA is bound to a column and the mixture is heated in small steps. At each step, the column is washed; sequences that melt become single-stranded and wash off the column. The temperatures at which labelled DNA comes off the column reflects the amount of similarity between sequences. These results are combined to determine the degree of genetic similarity between organisms. One method was introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single membrane. These samples would have to be separated in their own lanes inside the membranes and then the membrane would have to be rotated to a different angle where it would result in simultaneous hybridization with many different DNA probes.
DNA–DNA hybridization was once used as a primary method to distinguish bacterial species; a similarity value greater than 70% and ≤ 5 ºC in ΔTm in the stability of the heteroduplex is described as indicating that the compared strains belonged to distinct species. In 2014, a threshold of 79% similarity has been suggested to separate bacterial subspecies. DNA–DNA hybridization has not been tested much worldwide because it could take years to get results and it's not always that easy to perform in routine laboratories. However in 2004, there has been a new method tested out by digesting melting profiles with Sau3A in microplates in order to get a faster DNA–DNA hybridization test result.
In zoology
and Jon Ahlquist, pioneers of the technique, used DNA–DNA hybridization to examine the phylogenetic relationships of avians and primates.
In radioactivity
In 1969, one such method was performed by Mary Lou Pardue and Joseph G. Gall at the Yale University through radioactivity where it involved the hybridization of a radioactive test DNA in solution to the stationary DNA of a cytological preparation, which is identified as autoradiography.
Critics argue that the technique is inaccurate for comparison of closely related species, as any attempt to measure differences between orthologous sequences between organisms is overwhelmed by the hybridization of paralogous sequences within an organism's genome. DNA sequencing and computational comparisons of sequences is now generally the method for determining genetic distance, although the technique is still used in microbiology to help identify bacteria.
The modern approach is to carry out DNA–DNA hybridization in silico using completely or partially sequenced genomes. The developed at DSMZ is the most accurate known tool for calculating DDH-analogous values. Among other algorithmic improvements, it solves the problem with paralogous sequences by carefully filtering them from the matches between the two genome sequences.
