Method for the identification of essential and conditional essential genes

摘要

A method for identifying an essential gene of an organism, which method comprises: (i) providing a library of transposon insertion mutants of the said organism, wherein the transposon comprises an RNA polymerase recognition sequence; (ii) isolating chromosomal DNA from the library of (i), (iii) digesting the chromosomal DNA with a restriction endonuclease that is capable of cutting 5′ of the RNA polymerase recognition site in the transposon and 3′ of the RNA polymerase recognition site in the chromosomal DNA flanking the transposon; (iv) self-ligating the digested DNA, (v) amplifying the self-ligated DNA by inverse PCR (iPCR), (vi) transcribing RNA from the amplified DNA, (vii) hybridising the transcribed RNA with an oligonucleotide array; and (viii) identifying a probe on the oligonucleotide array which corresponds to an essential gene of the organism. Genes identified in such a method are useful as substrates for use in screens for antibacterials, antiparasitics, fungicides, pesticides and herbicides.