Patent ID: 6284468
Filing Date: 2001-09-04
Classification: C12N

Abstract:
A method for detecting the presence of initiation of transcription activity by RNA polymerase, comprising:(a) providing:(i) a test sample suspected of comprising RNA polymerase which specifically binds to a first nucleotide sequence;(ii) a restriction enzyme which binds to a second nucleotide sequence and which cleaves a DNA sequence at a restriction site;(iii) a recombinant plasmid comprising said first and second nucleotide sequences and said restriction site, wherein said restriction site is located at a position selected from being comprised within said first nucleotide sequence, being comprised within said second nucleotide sequence, being substantially adjacent to said first nucleotide sequence, and being substantially adjacent to said second nucleotide sequence; and(iv) a mixture of ribonucleotide triphosphates;(b) contacting said test sample with said recombinant plasmid to generate a contacted test sample, wherein said contacting is under conditions such that said RNA polymerase specifically binds to said first nucleotide sequence in said recombinant plasmid to form a bound plasmid;(c) treating said contacted test sample with said restriction enzyme to generate a treated test sample, wherein the treatment is under conditions such that said bound plasmid is not substantially cleaved by said restriction enzyme;(d) mixing said treated test sample with said mixture of ribonucleotide triphosphates to generate a mixed test sample; and(e) detecting the presence of uncleaved plasmid in said mixed test sample thereby detecting the presence of said initiation of transcription activity by said RNA polymerase in said test sample.