US 5,994,069 AGranted

Detection of nucleic acids by multiple sequential invasive cleavages

AssigneeThird Wave Technologies, Inc.

InventorsJeff G. Hall, Victor I. Lyamichev, Andrea L. Mast, Mary Ann D. Brow

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Abstract

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity 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. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

Claims34

Dependent claims

A method of detecting the presence of a target nucleic acid molecule comprising:

a) providing:
i) a cleavage means,
ii) a source of a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is downstream from said second region and wherein said second region is contiguous to and downstream from said third region;
iii) first and second oligonucleotides having 3' and 5' portions, wherein said 3' portion of said first oligonucleotide contains a sequence complementary to said third region of said first target nucleic acid and wherein said 5' portion of said first oligonucleotide and said 3' portion of said second oligonucleotide each contain sequence fully complementary to said second region of said first target nucleic acid, and wherein said 5' portion of said second oligonucleotide contains sequence complementary to said first region of said first target nucleic acid;
iv) a source of a second target nucleic acid, said second target nucleic acid having a first region, a second region and a third region, wherein said first region is located downstream from said second region and wherein said second region is contiguous to and downstream from said third region;
v) a third oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said third oligonucleotide contains a sequence fully complementary to said second region of said second target nucleic acid and wherein said 3' portion of said third oligonucleotide contains a sequence complementary to said third region of said second target nucleic acid;
b) generating a first cleavage structure wherein at least said 3' portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5' portion of said second oligonucleotide is annealed to said first target nucleic acid and wherein cleavage of said first cleavage structure occurs via said cleavage means thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fourth oligonucleotide contains a sequence complementary to said first region of said second target nucleic acid and wherein said 3' portion of said fourth oligonucleotide contains a sequence fully complementary to said second region of said second target nucleic acid;
c) generating a second cleavage structure under conditions wherein said at least said 3' portion of said third oligonucleotide is annealed to said second target nucleic acid and wherein at least said 5' portion of said fourth oligonucleotide is annealed to said second target nucleic acid oligonucleotide and wherein cleavage of said second cleavage structure occurs to generate a fifth oligonucleotide, said fifth oligonucleotide having a 3'-hydroxyl group;
and
d) detecting said fifth oligonucleotide, thereby detecting the presence of said first and second target nucleic acids.

7 more elements

A method of detecting the presence of a target nucleic acid molecule comprising:

a) providing:
i) a cleavage means,
ii) a source of a first target nucleic acid, said first target nucleic acid having a first region, a second region, a third region and a fourth region, wherein said first region is located downstream from said second region, said second region is contiguous to and downstream from said third region and said third region is located adjacent to and downstream from said fourth region;
iii) a first oligonucleotide complementary to said fourth region of said first target nucleic acid;
iv) second and third oligonucleotides having 3' and 5' portions, wherein said 3' portion of said second oligonucleotide contains a sequence complementary to said third region of said first target nucleic acid and wherein said 5' portion of said second oligonucleotide and said 3' portion of said third oligonucleotide each contain sequence fully complementary to said second region of said first target nucleic acid, and wherein said 5' portion of said third oligonucleotide contains sequence complementary to said first region of said first target nucleic acid;
iv) a source of a second target nucleic acid, said second target nucleic acid having a first region, a second region and a third region, wherein said first region is located downstream from said second region and wherein said second region is contiguous to and downstream from said third region;
v) a fourth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fourth oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid and wherein said 3' portion of said fourth oligonucleotide contains a sequence fully complementary to said third region of said second target nucleic acid;
b) generating a first cleavage structure wherein said first oligonucleotide is annealed to said fourth region of said first target nucleic acid and wherein at least said 3' portion of said second oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5' portion of said third oligonucleotide is annealed to said first target nucleic acid and wherein cleavage of said first cleavage structure occurs thereby cleaving said second oligonucleotide to generate a fifth oligonucleotide, said fifth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fifth oligonucleotide contains a sequence complementary to said first region of said second target nucleic acid and wherein said 3' portion of said fifth oligonucleotide contains a sequence fully complementary to said second region of said second target nucleic acid;
c) generating a second cleavage structure under conditions wherein at least said 3' portion of said fourth oligonucleotide is annealed to said second target nucleic acid and wherein at least said 5' portion of said fifth oligonucleotide is annealed to said second target nucleic acid and wherein cleavage of said second cleavage structure occurs to generate a sixth oligonucleotide, said sixth oligonucleotide having a 3'-hydroxyl group;
and
d) detecting said sixth oligonucleotide, thereby detecting the presence of said first and second target nucleic acids.

8 more elements

A method of detecting the presence of a target nucleic acid molecule comprising:

a) providing:
i) a cleavage means,
ii) a source of a target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is downstream from said second region and wherein said second region is contiguous to and downstream from said third region;
iii) first and second oligonucleotides having 3' and 5' portions, wherein said 3' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5' portion of said first oligonucleotide and said 3' portion of said second oligonucleotide each contain sequence fully complementary to said second region of said target nucleic acid, and wherein said 5' portion of said second oligonucleotide contains sequence complementary to said first region of said target nucleic acid;
iv) a third oligonucleotide having a 5' and a 3' portion wherein said 3' portion of said third oligonucleotide contains a sequence complementary to said 5' portion of said first oligonucleotide;
b) generating a first cleavage structure wherein at least said 3' portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5' portion of said second oligonucleotide is annealed to said target nucleic acid and wherein cleavage of said first cleavage structure occurs thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide having a first region, a second region and a third region, wherein said first region is located adjacent to and upstream of said second region and wherein said second region is located adjacent to and upstream of said third region and wherein said third region of said fourth oligonucleotide contains a region of self-complementarity;
c) generating a second cleavage structure under conditions wherein said at least said 3' portion of said third oligonucleotide is annealed to said first region of said fourth oligonucleotide and wherein said third region of said fourth oligonucleotide forms a hairpin structure and wherein cleavage of said second cleavage structure occurs to generate a fifth oligonucleotide, said fifth oligonucleotide having a 3'-hydroxyl group;
and
d) detecting said fifth oligonucleotide, thereby detecting the presence of said target nucleic acid.

6 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/823,516Filed 24 Mar 1997 · continuation in part
Priority: 24 Jan 1996
Granted: 30 Nov 1999
Term ends: Not estimatedThe source records don’t support an estimate.

Classification10 codes

C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]
C12N 9/1252
Nucleotidyltransferases: dNA-directed DNA polymerase , i.e. DNA replicase
C12Q 1/6816
Hybridisation assays: characterised by the detection means
C12Q 1/6823
Characterised by the detection means: release of bound markers

Family

Serial599,491Parent of this continuation-in-part · filed 24 Jan 1996
Serial682,853Parent of this continuation-in-part · filed 12 Jul 1996
Serial756,386Parent of this continuation-in-part · filed 26 Nov 1996
Serial759,038Parent of this continuation-in-part · filed 2 Dec 1996
Serial758,314Parent of this continuation-in-part · filed 2 Dec 1996
PCTUS9701072Parent of this continuation-in-part · filed 21 Jan 1997
US 5,994,069 AThis patent

From the US filing history. Foreign family members aren’t listed.