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Compositions and methods for determining the activity of DNA-binding proteins and of initiation of transcription

  • US 6,297,013 B1
  • Filed: 06/24/1999
  • Issued: 10/02/2001
  • Est. Priority Date: 06/24/1999
  • Status: Expired due to Fees
First Claim
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1. A method for detecting the presence of a sequence-specific DNA-binding molecule capable of specifically binding to a nucleotide sequence, comprising:

  • (a) providing;

    (i) a first sample suspected of containing a sequence-specific DNA-binding molecule capable of specifically binding to a first nucleotide sequence;

    (ii) a restriction enzyme capable of binding to a second nucleotide sequence and cleaving at a restriction site; and

    (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 within said first nucleotide sequence, within said second nucleotide sequence, substantially adjacent to said first nucleotide sequence, and substantially adjacent to said second nucleotide sequence; and

    (b) contacting said first sample with said recombinant plasmid to generate a contacted sample, under conditions such that said sequence-specific DNA-binding molecule specifically binds to said first nucleotide sequence in said recombinant plasmid to generate a bound plasmid;

    (c) treating said contacted sample with said restriction enzyme under conditions such that said bound plasmid is not substantially cleaved by said restriction enzyme; and

    (d) detecting the presence of uncleaved plasmid in said contacted sample, thereby detecting the presence of said sequence-specific DNA-binding molecule in said first sample.

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