# US 2008/0188375 A1 · Reactions on a solid surface

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2008/0188375 A1
- Document ID: US20080188375A1
- Status: Published application
- Kind: A1
- Application number: 11/926,120
- Filed: 2007-10-28
- Published: 2008-08-07
- Earliest claimed: 1996-01-24
- Assignees: Third Wave Technologies, Inc.
- Inventors: Bruce P. Neri; Jeff G. Hall; Victor I. Lyamichev; Lloyd M. Smith
- Web page: https://patentgrep.com/patent/US20080188375A1

## CPC

- C12Q 1/6823: Release of bound markers
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Q 1/6827: for detection of mutation or polymorphism
- Y10S 435/81: Packaged device or kit
- Y10S 435/822: using bacteria or actinomycetales
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## 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

### Claim 1

A system comprising oligonucleotides capable of hybridizing to a target nucleic acid to form an invasive cleavage structure, wherein said oligonucleotides comprise first and second oligonucleotides, said first oligonucleotide comprising a portion complementary to a first region of said target nucleic acid and said second oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′ portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid, a solid support, and a 5′ nuclease, wherein one or more of said oligonucleotides is attached to said solid support.

### Claim 2 (depends on 1)

The system of claim 1, wherein said 5′ nuclease comprises a FEN-1 endonuclease.

### Claim 3 (depends on 1)

The system of claim 1, wherein said 5′ nuclease comprises a polymerase.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 4)

The system of claim 4, wherein said first oligonucleotides further comprise a quencher molecule.

### Claim 6 (depends on 1)

The system of claim 1, further comprising a spacer molecule, wherein said one or more oligonucleotides is attached to said solid support through said spacer molecule.

### Claim 7 (depends on 6)

The system of claim 6, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

### Claim 8 (depends on 1)

The system of claim 1, wherein said solid support comprises a support selected from the group consisting of a glass solid support, a plastic solid support, a silicon solid support, a latex solid support and a hydrogel solid support.

### Claim 9 (depends on 1)

The system of claim 1, wherein said solid support comprises a solid support selected from the group consisting of a bead, a multi-well plate, a column, and a microarray.

### Claim 10 (depends on 1)

The system of claim 1, wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.

### Claim 11

A method for characterizing a nucleic acid comprising:
a) providing:
i. a sample suspected of containing a target nucleic acid;
ii. oligonucleotides capable of hybridizing to said target nucleic acid to form an invasive cleavage structure, wherein said oligonucleotides comprise first and second oligonucleotides, said first oligonucleotide comprising a portion complementary to a first region of said target nucleic acid and said second oligonucleotide comprising a 3′ portion and a 5′ portion, said 5′ portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid;
iii. a solid support, wherein one or more of said oligonucleotides is attached to said solid support; and
iv. an agent capable of detecting the presence of an invasive cleavage structure;
b) exposing said sample to said oligonucleotides and said agent, wherein said exposing said sample to said oligonucleotides and said agent comprises exposing said sample to said oligonucleotides and said agent under conditions wherein an invasive cleavage structure is formed between a target nucleic acid and said oligonucleotides if said target nucleic acid is present in said sample; and
c) detecting said invasive cleavage structure.

### Claim 12 (depends on 11)

The method of claim 11, wherein said agent comprises a 5′ nuclease.

### Claim 13 (depends on 12)

The method of claim 12, wherein said 5′ nuclease comprises a FEN-1 endonuclease.

### Claim 14 (depends on 12)

The method of claim 12, wherein said 5′ nuclease comprises a polymerase.

### Claim 15 (depends on 11)

The method of claim 11, wherein one or more of said first oligonucleotides and/or one or more of said second oligonucleotides is attached to said solid support.

### Claim 16 (depends on 11)

The method of claim 11, wherein said first oligonucleotides comprise a fluorescent dye.

### Claim 17 (depends on 16)

The method of claim 16, wherein said first oligonucleotides further comprise a quencher molecule.

### Claim 18 (depends on 11)

The method of claim 11, further providing a spacer molecule, wherein said one or more oligonucleotides is attached to said solid support through said spacer molecule.

### Claim 19 (depends on 18)

The method of claim 18, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

### Claim 20 (depends on 11)

The method of claim 11, wherein said solid support comprises a support selected from the group consisting of a glass solid support, a plastic solid support, a silicon solid support, a latex solid support and a hydrogel solid support.

### Claim 21 (depends on 11)

The method of claim 11, wherein said solid support comprises a solid support selected from the group consisting of a bead, a multi-well plate, a column, and a microarray.

### Claim 22 (depends on 11)

The method of claim 11, wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.

### Claim 23

A method for cleaving multiple oligonucleotides comprising:
a) providing:
i. a plurality of first oligonucleotides attached to a solid support;
ii. a second oligonucleotide attached to said solid support; and
iii. a cleavage agent;
wherein said first oligonucleotides comprise a portion complementary to a first region of a target nucleic acid; and wherein said second oligonucleotide comprises a 3′ portion and a 5′ portion, said 5′ portion complementary to a second region of said target nucleic acid downstream of and contiguous to said first portion of said target nucleic acid;
b) exposing said solid support to said cleavage agent under conditions such that:
i. a first cleavage structure is formed, said first cleavage structure comprising one of said first oligonucleotides and said second oligonucleotide;
ii. said cleavage agent cleaves said first oligonucleotide in said first cleavage structure to produce a first cleavage fragment;
iii. after cleavage of said first first oligonucleotide, a second cleavage structure is formed, said second cleavage structure comprising a second of said first oligonucleotides and said second oligonucleotide;
iv. said cleavage agent cleaves said first oligonucleotide in said second cleavage structure to produce a second cleavage fragment;
wherein said conditions comprise exposing a target nucleic acid to said solid support; and
c) detecting the presence of said first or said second cleavage fragments.

### Claim 24 (depends on 23)

The method of claim 23, wherein said cleavage agent comprises a structure-specific nuclease.

### Claim 25 (depends on 24)

The method of claim 24, wherein said cleavage agent comprises a 5′ nuclease.

### Claim 26 (depends on 25)

The method of claim 25, wherein said 5′ nuclease comprises a FEN-1 endonuclease.

### Claim 27 (depends on 25)

The method of claim 25, wherein said 5′ nuclease comprises a polymerase.

### Claim 28 (depends on 23)

The method of claim 23, wherein said second oligonucleotide is attached to said solid support through a spacer molecule.

### Claim 29 (depends on 28)

The method of claim 28, wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with three or more of said first oligonucleotides.

### Claim 30 (depends on 29)

The method of claim 29, wherein said spacer molecule is selected to allow said second oligonucleotide to form a cleavage structure with ten or more of said first oligonucleotides.

### Claim 31 (depends on 28)

The method of claim 28, wherein said spacer molecule is selected from the group consisting of a carbon chain, a polynucleotide, biotin, and a polyglycol.

### Claim 32 (depends on 23)

The method of claim 23, wherein said solid support comprises a support selected from the group consisting of a glass solid support, a plastic solid support, a silicon solid support, a latex solid support and a hydrogel solid support.

### Claim 33 (depends on 23)

The method of claim 23, wherein said solid support comprises a solid support selected from the group consisting of a bead, a multi-well plate, a column, and a microarray.

### Claim 34 (depends on 23)

The method of claim 23, wherein said solid support is coated with a material selected from the group consisting of gold and streptavidin.