Reactions on a solid surface

• US 7,527,928 B2
• Filed: 12/04/2002
• Issued: 05/05/2009
• Est. Priority Date: 11/29/1996
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Neri, Bruce P., Hall, Jeff G., Smith, Lloyd M., Lyamichev, Victor
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): CALAMITA, HEATHER
• Application Number:
  US10/309,584
• Publication Number:
  US 20040214174A1
• Time in Patent Office: 2,344 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]

Abstract

The present invention relates to compositions and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for forming a nucleic acid cleavage structure on a solid support and cleaving the nucleic acid cleavage structure in a site-specific manner. For example, in some embodiments, a 5′ nuclease activity from any of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

Claims

1. A system comprising a target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region, oligonucleotides capable of hybridizing to said target nucleic acid to form an invasive cleavage structure, wherein said oligonucleotides comprise first and second oligonucleotides, where at least a portion of said first oligonucleotide is completely complementary to said first region of said target nucleic acid, and wherein said second oligonucleotide comprises a 3′

portion and a 5′

portion, wherein said 5′

portion is completely complementary to said second region of said target nucleic acid, a solid support, and a thermostable archaeal FEN-1 endonuclease, wherein said FEN-1 is active at 55°

C. and above, for detecting the presence of an invasive cleavage structure;

wherein one or more of said oligonucleotides is attached to said solid support, and wherein an invasive cleavage structure forms when a first and a second oligonucleotide hybridize to said first and second regions of said target nucleic acid.

2. The system of claim 1, wherein one or more of said first oligonucleotides is attached to said solid support.

3. The system of claim 1, wherein one or more of said second oligonucleotides is attached to said solid support.

4. The system of claim 1, wherein one or more of said first oligonucleotides and one or more of said second oligonucleotides is attached to said solid support.

5. The system of claim 1, wherein said first oligonucleotides comprise signal probe oligonucleotides.

6. The system of claim 1, wherein said first oligonucleotides comprise a fluorescent dye.

7. The system of claim 6, wherein said first oligonucleotides further comprise a quencher molecule.

8. The system of claim 1, wherein said target nucleic acid is attached to said solid support.

9. The system of claim 1, wherein said solid support comprises a bead.