Solid-phase reactions

• US 7,485,443 B2
• Filed: 07/17/2002
• Issued: 02/03/2009
• Est. Priority Date: 07/17/2001
• Status: Expired due to Fees

• Current Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Original Assignee: Northwestern University
• Inventors: Kelso, David, Hall, Jeff, Stevens, Priscilla Wilkins, Smith, Lloyd, Neri, Bruce P., Lyamichev, Victor, Kwiatkowski, Robert W. Jr.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Wilder; Cynthia B
• Application Number:
  US10/197,189
• Publication Number:
  US 20030119020A1
• Time in Patent Office: 2,393 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.21, 536/23.1, 536/24.3, 536/24.32, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/549 characterised by the capture oligonucleotide being a reporter labelled capture oligonucleotide

Abstract

The present invention relates to systems, 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 attaching nucleic acids to solid supports and modifying nucleic acids. For example, in some embodiments, the 5′ nuclease activity of a cleavage agent is used to cleave a cleavage structure formed on the solid support, the occurrence of the cleavage event indicating the presence of specific nucleic acid sequences.

Claims

1. A method for detecting polymorphisms in unamplified DNA, comprising:

a) providing;

i) unamplified DNA, wherein said unamplified DNA comprises 50 micrograms or less of genomic DNA wherein said genomic DNA has not been subjected to an in vitro amplification method;

ii) reagents for conducting an invasive cleavage reaction, wherein said reagents comprise a plurality of oligonucleotide pairs, wherein each of said oligonucleotide pairs comprises a probe oligonucleotide and an upstream oligonucleotide, wherein said probe oligonucleotide associates and dissociates from a target sequence, wherein said upstream oligonucleotide is configured to hybridize to a target sequence in a manner that overlaps with said probe oligonucleotide, wherein said probe and upstream oligonucleotides are attached to the same solid surface, wherein said upstream oligonucleotide is attached to said surface via a spacer molecule that has a length of 10 hexaethylene molecules, and wherein the ratio of probe oligonucleotide to upstream oligonucleotide is from 5;

1 to 30;

1; and

b) treating said unamplified DNA with said reagents under conditions such that invasive cleavage reactions occur on said solid surface with said unamplified DNA and said reagents such that the presence or absence of a plurality of different polymorphisms in said genomic DNA are identified, and wherein said treating step is carried out in a single reaction vessel.

2. The method of claim 1, wherein said plurality of different polymorphisms comprises at least 1000 different polymorphisms.

3. The method of claim 1, wherein said plurality of different polymorphisms comprises at least 1,000,000 different polymorphisms.

4. The method of claim 1, wherein said solid surface comprises a microsphere.

5. The method of claim 1, wherein said solid surface comprises a plurality of microspheres.