The one-hybrid variation of this technique is designed to investigate protein–DNA interactions and uses a single fusion protein in which the AD is linked directly to the binding domain. The binding domain in this case however is not necessarily of fixed sequence as in two-hybrid protein–protein analysis but may be constituted by a library. This library can be selected against the desired target sequence, which is inserted in the promoter region of the reporter gene construct. In a positive-selection system, a binding domain that successfully binds the UAS and allows transcription is thus selected.
Note that selection of DNA-binding domains is not necessarily performed using a one-hybrid system, but may also be performed using a two-hybrid system in which the binding domain is varied and the bait and prey proteins are kept constant.