Compositions and methods for determining the activity of DNA-binding proteins and of initiation of transcription
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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Abstract
This invention provides plasmids that are useful in detecting and determining the DNA-binding activity of sequence-specific DNA-binding molecules. The invention further provides plasmids that are useful in detecting and determining the activity of KNA polymerases in initiating transcription. In particular, the invention relates to plasmids that contain unique restriction sites and cognate nucleotide recognition sequences for sequence-specific DNA-binding molecules. Also provided are methods for using the plasmids disclosed herein.
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Citations
12 Claims
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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:
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(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. - View Dependent Claims (2, 3)
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4. A method for determining DNA-binding activity of a sequence-specific DNA-binding molecule, comprising:
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(a) providing;
(i) a test sample suspected of comprising a sequence-specific DNA-binding molecule capable of specifically binding to a first nucleotide sequence;
(ii) a plurality of samples comprising known amounts of said sequence-specific DNA-binding molecule;
(iii) a restriction enzyme capable of binding to a second nucleotide sequence and of cleaving a DNA sequence at a restriction site; and
(iv) 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;
(b) contacting said test sample with said recombinant plasmid to generate a contacted test sample and contacting said plurality of samples with said recombinant plasmid to generate a contacted plurality of samples, wherein said contacting is under conditions such that said sequence-specific DNA-binding molecule 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 and treating said contacted plurality of samples with said restriction enzyme to generate a treated plurality of samples, wherein the treatment is under conditions such that said bound plasmid is not substantially cleaved by said restriction enzyme; and
(d) comparing the amount of uncleaved plasmid in said treated test sample with the amount of uncleaved plasmid in said treated plurality of samples, thereby determining DNA-binding activity of said sequence-specific DNA-binding molecule in said test sample. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12)
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Specification