The invention relates to methods of detecting nucleic acids, including methods of detecting one or more target
nucleic acid sequences in
multiplex branched-chain
DNA assays, are provided. Nucleic acids captured on a
solid support or suspending cells are detected, for example, through cooperative hybridization events that result in specific association of a
label with the nucleic acids. The invention further relates to methods to improve probe hybridization specificity and their application in
genotyping. The invention also relates to in situ detection of mis-joined
nucleic acid sequences. The invention relates to reducing false positive signals and improve
signal-to-background ratio in hybridization-based
nucleic acid detection
assay. The invention further relates to method to improve specificity in hybridization based nucleic acid using co-location probes. Compositions, tissue slides, sample of suspended cells, kits, and systems related to the methods are also described.