Methods for mutation detection
The invention relates to methods for detecting a mutation in a nucleic acid. Methods of the invention are useful for detecting and identifying mutations that are indicative of disease or the predisposition for disease.
1 . A method for detecting a mutation in a target nucleic acid, the method comprising the steps of:
(a) exposing an individually-optically detectable target nucleic acid template suspected to contain a mutation to a primer capable of hybridizing to a known region proximate to said mutation to form a target/primer duplex;
(b) exposing said duplex to one or more labeled nucleotides in the presence of a polymerase;
(c) incorporating one or more labeled nucleotide complementary to said target into said primer downstream of the duplex;
(d) identifying the incorporated labeled nucleotide; and
(e) repeating steps (b)-(d), thereby to determine if the mutation is present in said target.
2 . The method of claim 1 , wherein said primer is upstream of said mutation.
3 . The method of claim 1 , wherein said target is bound to a support.
4 . The method of claim 3 , wherein said support is glass.
5 . The method of claim 4 , wherein said glass has an epoxide coating thereon.
6 . The method of claim 5 , wherein said target is attached directly via an amine linkage.
7 . The method of claim 5 , wherein said target is attached via a linker pair.
8 . The method of claim 7 , wherein said linker pair is selected from biotin/avidin, antigen/antibody, and receptor/ligand.
9 . The method of claim 1 , the method further comprising the step of:
(f) shearing the target prior to step (a).
10 . The method of claim 9 , the method further comprising the step of:
(g) digesting the target.
11 . The method of claim 1 , wherein a 5′ end of a hybridized primer is between about 1 base and about 20 bases from the site suspected to contain a mutation.
12 . The method of claim 1 , wherein each of a plurality of targets is bound to a support.
13 . The method of claim 1 , wherein said polymerase is selected from the group consisting of Klenow, Nine degrees north, Vent, Taq, Tgo, sequenase, or any combination thereof.
14 . The method of claim 1 , wherein said nucleotide further comprises a removable blocking group attached to the 3′ hydroxyl.
15 . The method of claim 1 , wherein said target is exposed to a plurality of different nucleotide species, each comprising a different detectable label.
16 . The method of claim 1 , wherein said labeled nucleotide comprises an optically-detectable label.
17 . The method of claim 16 , wherein said optically-detectable label is a fluorescent label.
18 . The method of claim 17 , wherein said identifying step comprises exposing the incorporated labeled nucleotide to light that excites said fluorescent label.
19 . The method of claim 1 , wherein said incorporated labeled nucleotide is individually optically resolvable.
20 . A method for detecting a mutation in a target nucleic acid, the method comprising the steps of:
(a) exposing a target nucleic acid template suspected to contain a mutation to a primer capable of hybridizing to a known region proximate to said mutation;
(b) extending said primer, in the presence of at least one nucleotide, through a site suspected to contain said mutation in the presence of a polymerase;
(c) detaching the primer from said target;
(d) hybridizing a complement to the detached primer to form a duplex, wherein said duplex is individually-optically detectable;
(e) exposing said complement to at least one labeled nucleotide;
(f) identifying the incorporated labeled nucleotide; and
(g) repeating steps (e)-(f), thereby to detect if the mutation is present in said target.
21 . The method of claim 20 , wherein said primer is upstream of said mutation.
22 . The method of claim 20 , wherein each of the at least one nucleotides are unlabeled.
23 . The method of claim 20 , wherein the target is bound to a support.
24 . The method of claim 23 , wherein said support is glass.
25 . The method of claim 24 , wherein said glass has an epoxide coating thereon.
26 . The method of claim 25 , wherein said target is attached directly via an amine linkage.
27 . The method of claim 25 , wherein said target is attached via a linker pair.
28 . The method of claim 27 , wherein said linker pair is selected from biotin/avidin, antigen/antibody, and receptor/ligand.
29 . The method of claim 20 , wherein each of a plurality of targets is bound to a support.
30 . The method of claim 20 , wherein a 5′ end of a hybridized primer is between about 1 base and about 20 bases from the site suspected to contain a mutation.
31 . The method of claim 20 , the method further comprising the step of:
(h) exposing said complement to a plurality of chain terminating nucleotides.
32 . The method of claim 20 , wherein said polymerase is selected from the group consisting of Klenow, Nine degrees north, Vent, Taq, Tgo, sequenase, or any combination thereof.
33 . The method of claim 20 , wherein said nucleotide further comprises a removable blocking group attached to the 3′ hydroxyl.
34 . The method of claim 20 , wherein said target is exposed to a plurality of different nucleotide species, each comprising a different detectable label.
35 . The method of claim 20 , wherein said incorporated labeled nucleotide comprises an optically-detectable label.
36 . The method of claim 35 , wherein said optically-detectable label is a fluorescent label.
37 . The method of claim 36 , wherein said identifying step comprises exposing the incorporated labeled nucleotide to light that excites said fluorescent label.
38 . The method of claim 20 , wherein said incorporated labeled nucleotide is individually optically resolvable.