Nucleic acid analysis methods conducted in small reaction volumes
The present invention relates to reactions conducted on or in solid surface materials, including liquid phase reactions within capillary tubes and arrays of capillary tubes or open end reaction microchambers. A wide array of biomolecular and combinatorial synthesis reactions may be used with the present invention. For example, the present invention provides methods for forming and cleaving nucleic acid cleavage structures on or in solid surface materials.
1 . A method for detecting the presence of a target nucleic acid in a sample, comprising:
a) providing a microchamber containing a liquid reaction mixture, said liquid reaction mixture comprising:
i) a sample suspected of containing a target nucleic acid, and
ii) detection assay reagents comprising at least one oligonucleotide configured to form a cleavage structure with said target nucleic acid, a cleavage means capable of cleaving a cleavage structure, wherein said detection assay reagents are configured to produce a detectable signal upon cleavage of a cleavage structure;
b) incubating said microchamber under conditions that permit said cleavage reagents to produce a detectable signal; and
c) detecting said detectable signal, thereby detecting said target nucleic acid in said sample.
2 . The method of claim 1 , wherein said microchamber comprises a capillary tube.
3 . The method of claim 1 , wherein said detection assay reagents comprise reagents for conducting an invasive cleavage reaction.
4 . The method of claim 1 , wherein said detection assay reagents comprise a ligase.
5 . The method of claim 1 , wherein said sample comprises a blood sample.
6 . The method of claim 1 , wherein said sample is derived from a blood sample.
7 . The method of claim 1 , wherein said microchamber comprises a coating.
8 . The method of claim 7 , wherein said coating is selected from the group consisting of C-18, diol, PEG, cyclodextrine, PVA, polystyrene.
9 . The method of claim 1 , wherein said microchamber comprises a blocking agent.
10 . The method of claim 2 , wherein said providing comprises:
a) preparing said capillary tube to contain said detection assay reagents in dried form within said capillary tube; and
b) adding said sample suspected of containing a target nucleic acid in liquid form to said capillary tube under conditions such that said liquid reaction mixture is formed.
11 . The method of claim 2 , wherein said providing comprises:
a) preparing said capillary tube to contain said sample suspected of containing a target nucleic, in dried form within said capillary tube; and
b) adding said detection assay reagents in liquid form to said capillary tube under conditions such that said liquid reaction mixture is formed.
12 . A method for detecting the presence of one or more target nucleic acids in a sample, comprising:
a) providing a plurality of microchambers, each microchamber containing a liquid reaction mixture, each said liquid reaction mixture comprising:
i) a sample suspected of containing at least one target nucleic acid, and
ii) detection assay reagents comprising at least one oligonucleotide configured to form a cleavage structure with a target nucleic acid, a cleavage means capable of cleaving a cleavage structure, wherein said detection assay reagents are configured to produce a detectable signal upon cleavage of a cleavage structure;
b) wherein at least two of said plurality of microchambers are configured to detect at least two different target nucleic acids,
c) incubating said plurality of microchambers under conditions that permit said cleavage reagents to produce detectable signal; and
d) detecting said detectable signal, thereby detecting said one or more target nucleic acids in said sample.
13 . The method of claim 12 , wherein said detection assay reagents comprise reagents for conducting an invasive cleavage reaction.
14 . The method of claim 12 wherein said detection assay reagents comprise a ligase.
15 . The method of claim 12 , wherein said microchamber comprises a capillary tube.
16 . The method of claim 15 , wherein said providing comprises:
a) preparing said capillary tube to contain said detection assay reagents in dried form within said capillary tube; and
b) adding said sample suspected of containing a target nucleic acid in liquid form to said capillary tube under conditions such that said liquid reaction mixture is formed.
17 . The method of claim 15 , wherein said providing comprises:
a) preparing said capillary tube to contain said sample suspected of containing a target nucleic, in dried form within said capillary tube; and
b) adding said detection assay reagents in liquid form to said capillary tube under conditions such that said liquid reaction mixture is formed.
18 . A kit comprising a plurality of capillary tubes comprising dried detection assay reagents configured to detect target nucleic acids, said dried detection assay reagents comprising reagents for conducting an invasive cleavage reaction.
19 . The kit of claim 18 , wherein one or more of said capillary tubes having detection assay reagents is configured to a target nucleic acid that at least one other capillary tube in said plurality of capillary tubes is not configured to detect.
20 . The kit of claim 18 , wherein each of said plurality of capillary tubes comprises detection assay reagents configured to detect a different target nucleic acid.
21 . The kit of claim 19 , wherein each of said plurality of capillary tubes further comprises detection assay reagents configured to detect the same target nucleic acid.
22 . The kit of claim 20 , wherein each of said plurality of capillary tubes further comprises detection assay reagents configured to detect the same target nucleic acid.