Rapid and Sensitive Detection of Nucleic Acid Sequence Variations

The ability to easily detect small mutations in nucleic acids, such as single base substitutions, can provide a powerful tool for use in cancer detection, perinatal screens for inherited diseases, and analysis of genetic polymorphisms such as genetic mapping or for identification purposes. Current approaches make use of the mismatch that occurs between complimentary strands of DNA when there is a genetic mutation, the electrophoretic mobility differences caused by small sequence changes, and chemicals or enzymes that can cleave heteroduplex sites. Some of these methods, however, prove to be too cumbersome, are unable to pinpoint mutations, only detect a subset of mutations, or involve the use of hazardous materials.

The current invention takes advantage of the ability of transposons, or mobile genetic elements, to move from one part of the genome to another by the cleavage and joining of their sequences into the target site; a reaction facilitated by a transposase enzyme. The phage Mu transposase is capable of inserting the right end sequence of the Mu transposon into any DNA sequence both in vitro and in vivo. The surprising discovery that the Mu transposase displays a strong preference for inserting Mu-end DNA into mismatched sites, the very sites which occur when DNA is mutated and paired with its complimentary strand that does not have the corresponding mutation, makes it a powerful tool for detecting variations in nucleic acid sequences. In this system, the transposition of Mu-end DNA at a site is used to indicate the presence of a nucleic acid mismatch or mutation at that site. The invention can be used with labeled Mu-end DNA to further facilitate the precise mapping of the mutations. This specificity allows Mu to detect even single base mutations amongst a large quantity of non-specific DNA. The Mu detection system is simple, rapid, and highly sensitive compared to current methods and can find a broad range of use in genetic research and the diagnosis of several diseases such as cystic fibrosis, spinal and bulbar muscular dystrophy, human fragile-X syndrome and Huntington’s disease.