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Target-dependent transcription using deletion mutants of N4 RNA polymerase

  • US 20040191812A1
  • Filed: 12/23/2003
  • Published: 09/30/2004
  • Est. Priority Date: 05/22/2001
  • Status: Active Grant
First Claim
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1. ) A method for detecting a target nucleic acid sequence, the method comprising:

  • a) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′

    -end of a first target-complementary sequence is complementary to the 5′

    -end of the target nucleic acid sequence, and wherein the 3′

    -end of a second target-complementary sequence is complementary to the 3′

    -end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3′

    - of the first target-complementary sequence, wherein the promoter binds an RNA polymerase that lacks helicase-like activity and that can transcribe RNA using a single-stranded promoter;

    b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form a target probe-target complex;

    c) contacting the target probe-target complex with a ligase under ligation conditions to form a transcription substrate;

    d) contacting the transcription substrate with the RNA polymerase;

    e) optionally, repeating steps (a) through (e); and

    f) detecting the transcription product.

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