Detection of ras mutations
First Claim
Patent Images
1. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67.
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0 Petitions
Accused Products
Abstract
Mutations in K-ras, N-ras, and H-ras were determined using target specific primers and probes in REMS-PCR methods, nested PCR methods employing a restriction endonuclease, and REMS-PCR methods using molecular beacons.
43 Citations
37 Claims
-
1. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67.
- 5, SEQ ID NO;
-
2. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, or SEQ ID NO;
9.
- 5, SEQ ID NO;
-
3. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 10, SEQ ID NO;
12, or SEQ ID NO;
13.
- 10, SEQ ID NO;
-
4. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 14, SEQ ID NO;
15, SEQ ID NO;
16, or SEQ ID NO;
17. - View Dependent Claims (9, 10, 11)
- 14, SEQ ID NO;
-
5. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 18, SEQ ID NO;
19, SEQ ID NO;
20, or SEQ ID NO;
21.
- 18, SEQ ID NO;
-
6. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, or SEQ ID NO;
47.
- 22, SEQ ID NO;
-
7. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, or SEQ ID NO;
55.
- 48, SEQ ID NO;
-
8. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67. - View Dependent Claims (15, 16, 17)
- 56, SEQ ID NO;
-
12. One or more oligonucleotides comprising a sequence selected from SEQ ID NO:
- 19, SEQ ID NO;
20, or SEQ ID NO;
21, said one or more oligonucleotides comprising one or more fluorescence moieties and one or more fluorescence quenching moieties. - View Dependent Claims (13, 14)
- 19, SEQ ID NO;
-
18. A method for amplifying DNA comprising a mutant ras sequence in a sample comprising the steps of:
-
(B) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(iii) at least four different nucleoside triphosphates, one or more thermostable polymerases, and at least one thermostable restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both; and
(B) subjecting the admixture to one or more cycles of heating and cooling.
-
-
19. A method for determining one or more ras mutations in a DNA sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(iii) at least four different nucleoside triphosphates, one or more thermostable polymerases, and at least one thermostable restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both; and
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) separating the DNA by electrophoresis; and
(D) detecting the DNA comprising-a mutant ras sequence separated by electrophoresis in step (C).
-
-
20. A method for determining one or more ras mutations in a DNA sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(ii) at least four different nucleoside triphosphates, one or more thermostable polymerases, and at least one thermostable restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both; and
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) combining the admixture comprising amplified DNA with one or more immobilized oligonucleotides or one or more oligonucleotides capable of being immobilized, said one or more oligonucleotides being capable of hybridizing to DNA comprising a mutant ras sequence thereby capturing DNA comprising a mutant ras sequence; and
(D) detecting the captured DNA comprising a mutant ras sequence. - View Dependent Claims (21)
-
-
22. A method for determining one or more ras mutations in a DNA sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(iii) at least four different nucleoside triphosphates, one or more thermostable polymerases, and at least one thermostable restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both, and (iv) one or more oligonucleotides comprising one or more fluorescence moieties and one or more fluorescence quenching moieties, said one or more oligonucleotides being capable of hybridizing to DNA comprising a mutant ras sequence and capable of producing detectable fluorescence when hybridized thereto;
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) detecting the fluorescence. - View Dependent Claims (23, 24)
-
-
25. A method for amplifying DNA comprising a mutant ras sequence in a sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(iii) at least four different nucleoside triphosphates and one or more thermostable polymerases;
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) combining the admixture with one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NC;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67, provided at least one of the primers is different from the primers in step (A);
(D) subjecting the admixture to one or more cycles of heating and cooling; and
(E) combining the admixture with at least one restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both.
-
-
26. A method for determining one or more ras mutations in a DNA sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(iii) at least four different nucleoside triphosphates and one or more thermostable polymerases;
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) combining the admixture with one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67, provided at least one of the orimers is different from the primers in step (A);
(D) subjecting the admixture to one or more cycles of heating and cooling; and
(E) combining the admixture with at least one restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both;
(F) separating the DNA by electrophoresis; and
(G) detecting the DNA comprising a mutant ras sequence separated by electrophoresis in step (F).
-
-
27. A method for determining one or more ras mutations in a DNA sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(iii) at least four different nucleoside triphosphates and one or more thermostable polymerases;
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) combining the admixture with one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67, provided at least one of the primers is different from the primers in step (A);
(D) subjecting the admixture to one or more cycles of heating and cooling; and
(E) combining the admixture with at least one restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both;
(F) combining the admixture comprising amplified DNA with one or more immobilized oligonucleotides or oligonucleotides capable of being immobilized, said oligonucleotides being capable of hybridizing to DNA comprising a mutant ras sequence thereby capturing DNA comprising a mutant ras sequence; and
(G) detecting the captured DNA comprising a mutant ras sequence.
-
-
28. A method for determining one or more ras mutations in a DNA sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
30, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67,(iii) at least four different nucleoside triphosphates, and one or more thermostable polymerases;
(iv) one or more oligonucleotides comprising one or more fluorescence moieties and one or more fluorescence quenching moieties, said one or more oligonucleotides being capable of hybridizing to DNA comprising a mutant ras sequence and capable of producing detectable fluorescence when hybridized thereto;
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) combining the admixture produced in step (B) with one or more primer pairs selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, SEQ ID NO;
55, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67, provided at least one of the primers is different from the primers in step (A);
(D) subjecting the admixture to one or more cycles of heating and cooling;
(E) combining the admixture with at least one restriction endonuclease that is capable of directly cleaving wild type K-, H-, or N-ras sequence or cleaving a primer induced cleavage site, or both; and
(F) detecting the fluorescence. - View Dependent Claims (29, 30)
-
-
31. A kit comprising in one or more containers:
-
(i) one or more oligonucleotides comprising a sequence selected from SEQ ID NO;
5, SEQ ID NO;
6, SEQ ID NO;
7, SEQ ID NO;
8, SEQ ID NO;
9, SEQ ID NO;
10, SEQ ID NO;
12, SEQ ID NO;
13, SEQ ID NO;
14, SEQ ID NO;
15, SEQ ID NO;
16, SEQ ID NO;
17, SEQ ID NO;
18, SEQ ID NO;
22, SEQ ID NO;
23, SEQ ID NO;
24, SEQ ID NO;
25, SEQ ID NO;
26, SEQ ID NO;
27, SEQ ID NO;
28, SEQ ID NO;
29, SEQ ID NO;
30, SEQ ID NO;
31, SEQ ID NO;
32, SEQ ID NO;
33, SEQ ID NO;
34, SEQ ID NO;
35, SEQ ID NO;
36, SEQ ID NO;
37, SEQ ID NO;
38, SEQ ID NO;
39, SEQ ID NO;
40, SEQ ID NO;
41, SEQ ID NO;
42, SEQ ID NO;
43, SEQ ID NO;
44, SEQ ID NO;
45, SEQ ID NO;
46, SEQ ID NO;
47, SEQ ID NO;
48, SEQ ID NO;
49, SEQ ID NO;
50, SEQ ID NO;
51, SEQ ID NO;
52, SEQ ID NO;
53, SEQ ID NO;
54, or SEQ ID NO;
55;
(ii) one or more oligonucleotides selected from SEQ ID NO;
19, SEQ ID NO;
20, SEQ ID NO;
21, SEQ ID NO;
56, SEQ ID NO;
57, SEQ ID NO;
58, SEQ ID NO;
59, SEQ ID NO;
60, SEQ ID NO;
61, SEQ ID NO;
62, SEQ ID NO;
63, SEQ ID NO;
64, SEQ ID NO;
65, SEQ ID NO;
66, or SEQ ID NO;
67, the oligonucleotide or oligonucleotides comprising one or more fluorescence moieties and one or more fluorescence quenching moieties. - View Dependent Claims (32, 33, 34)
-
-
35. A method for amplifying and determining one or more target mutant sequences in a DNA sample comprising the steps of:
-
(A) forming an admixture comprising (i) the sample, (ii) one or more primer pairs specific for one or more of the mutant target sequences, (iii) at least four different nucleoside triphosphates, (iv) one or more thermostable polymerases, (v) at least one thermostable restriction endonuclease that is capable of directly cleaving wild type DNA sequence of the mutant target or mutant targets or cleaving a primer induced cleavage site, or both, (vi) one or more oligonucleotides comprising one or more fluorescence moieties and one or more fluorescence quenching moieties, said one or more oligonucleotides being capable of hybridizing to DNA comprising the target mutant sequence or target mutant sequences and capable of producing detectable fluorescence when hybridized thereto;
(B) subjecting the admixture to one or more cycles of heating and cooling;
(C) detecting the fluorescence. - View Dependent Claims (36, 37)
-
Specification