Enzymatic nucleic acid synthesis: methods for direct detection of tagged monomers
2 Assignments
0 Petitions
Accused Products
Abstract
Nucleotide triphosphate probes containing a molecular and/or atomic tag on a a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.
-
Citations
136 Claims
-
1. (canceled)
-
2. (canceled)
-
3. (canceled)
-
4. (canceled)
-
5. (canceled)
-
6. (canceled)
-
7. (canceled)
-
8. (canceled)
-
9. (canceled)
-
10. (canceled)
-
11. (canceled)
-
12. (canceled)
-
13. (canceled)
-
14. A method for sequencing DNA samples comprising the steps of:
-
amplifying a DNA sample;
separating the amplified DNA sample into two single stranded DNA sequences, removing one of the two single stranded DNA sequences, hybridizing the remaining single stranded DNA sequence with a primer, preparing four aliquots of the hybridized DNA sequence, subjecting each aliquot to a polymerase reaction in the presence of a different nucleotide type including an unique molecular and/or atomic tag on a β and
/or γ
phosphate group thereof, where the tag alters fidelity of nucleotide incorporation and decreases or eliminates mis-incorporation,detecting an extent of released tagged pyrophosphate for each aliquot, and comparing the extents of the released tagged pyrophosphates to identify the tagged nucleotide type corresponding to the target position in the hybridized DNA sequence. - View Dependent Claims (21, 22, 23, 24, 25, 26, 45)
-
-
15. (canceled)
-
16. (canceled)
-
17. (canceled)
-
18. (canceled)
-
19. (canceled)
-
20. (canceled)
-
27. A method for sequencing DNA comprising the steps:
-
amplifying a sample DNA, separating the amplified DNA into two single stranded DNA sequences, removing one of the two single stranded DNA sequences, hybridizing the remaining single stranded DNA sequence with a primer, first exposing the hybridized DNA sequence to a first polymerase reaction in the presence of a first nucleotide type including a molecular and/or atomic tag on a β and
/or γ
phosphate group thereof, where the tag alters fidelity of nucleotide incorporation and decreases or eliminates mis-incorporation,detecting an extent of released tagged pyrophosphate, if the extent of released tagged pyrophosphate evidences nucleotide type incorporation, then identifying the incorporated nucleotide type and repeating the first exposing step, if the extent of released tagged pyrophosphate evidences no nucleotide incorporation, then adding a nucleotide-degrading enzyme to degrade the first nucleotide type and after degradation, removing the reaction medium, second exposing the hybridized DNA sequence to a second polymerase reaction in the presence of a different nucleotide type including a molecular and/or atomic tag on a β and
/or γ
phosphate group thereof, where the tag alters fidelity of nucleotide incorporation and decreases or eliminates mis-incorporation,repeating the previous four steps until the nucleotide type corresponding to a target position of the hybridized DNA sequence is identified. - View Dependent Claims (28, 29, 30, 31, 32)
-
-
33. A method for sequencing nucleic acids comprising the steps of:
-
immobilizing a single stranded DNA sequence on a support, hybridizing the single stranded DNA sequence with a primer, subjecting the hybridized single stranded DNA sequence to a polymerase reaction in the presence of a first nucleotide type including a molecular and/or atomic tag on a β and
/or γ
phosphate group thereof, where the tag alters fidelity of base incorporation and decreases or eliminates mis-incorporation,detecting an extent of released tagged pyrophosphate, adding a nucleotide degrading enzyme to the polymerase reaction including the first nucleotide type, repeating the subjecting, detecting and adding steps, until the nucleotide type corresponding to a target position of the hybridized DNA sequence is identified. - View Dependent Claims (34, 35, 136, 37, 38)
-
-
39. A method for sequencing nucleic acids comprising the steps of:
-
immobilizing a single stranded DNA sequence of the amplified DNA sample on a support, hybridizing the immobilized single stranded DNA sequence with a primer, subjecting the hybridized single stranded DNA sequence to a polymerase reaction containing beta-phosphate and/or gamma-phosphate tagged nucleotides, where each nucleotide is a 3′
-protected, 2′
deoxynucleotide and each nucleotide type contains a different tag with a different detectable property and where the tag alters fidelity of base incorporation and decreases or eliminates mis-incorporation,determining if tagged pyrophosphate is released and detecting the detectable property of the tag to determine an identity of the incorporated nucleotide, removing the 3′
-blocking group to permit another 3′
-protected-2′
deoxynucleotide to be incorporated,repeating the subjecting, determining, and removing steps, until a sequence of nucleotide of the DNA sample is determined. - View Dependent Claims (40)
-
-
41. A method for sequencing nucleic acids comprising the steps of:
-
amplifying a DNA sample, immobilizing a single stranded DNA sequence of the DNA sample on a support, hybridizing the single stranded DNA sequence with a primer, subjecting the hybridized single stranded DNA sequence to a polymerase reaction containing beta- and/or gamma-tagged nucleotides, where each nucleotide is a 3′
-protected 2′
deoxynucleotide and each nucleotide type contains a different tag with a different detectable property,determining if tagged pyrophosphate is released and detecting the detectable property of the tag to determine the identity of the incorporated nucleotide, removing the 3′
-blocking group to permit a further 3′
-protected-2′
deoxynucleotide to be incorporated,repeating the subjecting, determining, and removing steps, until the sequence of the DNA sequence is determined. - View Dependent Claims (42)
-
-
43. A method for sequencing a nucleic acid by directly detecting the tag attached to the released, tagged pyrophosphate when a nucleotide analog is incorporated into a growing strand of DNA in a polymerase reaction, which comprises the following steps:
-
amplifying a sample DNA;
immobilizing single stranded DNA sequence on a support;
introducing a primer onto the single stranded DNA sequence;
subjecting the primed DNA to a polymerase reaction containing one or more different nucleotide analogs containing a tag attached to the beta and/or gamma phosphate such that the nucleotide analog is incorporated into the growing strand of DNA and wherein the incorporated nucleotide analog terminates the polymerase reaction due to the presence of a 3′
-protecting group on the nucleotide;
detecting the tagged pyrophosphate that is released as a result of analog incorporation into the growing DNA strand, so as to identify the incorporated nucleotide analog;
cleaving the 3′
-protecting group from the nucleotide analog that was incorporated into the growing strand of DNA;
repeating the steps of subjecting, detecting and cleaving until the sequence of the DNA is determined.
-
-
44. A method of identifying a base at a target position in a single-stranded sample DNA sequence wherein an extension primer, which hybridizes to the sample DNA immediately adjacent to the target position is provided and the sample DNA and extension primer are subjected to a polymerase reaction in the presence of a deoxynucleotide or 3′
- -protected 2′
deoxynucleotide whereby the deoxynucleotide or 3′
-protected 2′
deoxynucleotide is incorporated and releases tagged pyrophosphate if it is complementary to the base in the target position, and whereby any release of tagged pyrophosphate is directly detected, different deoxynucleotides or 3′
-protected 2′
deoxynucleotide being added either to separate aliquots of sample-primer mixture or successively to the same sample-primer mixture and subjected to the polymerase reaction to indicate which deoxynucleotide or 3′
-protected 2′
deoxynucleotide is incorporated by directly detecting the tagged pyrophosphate and wherein released tagged pyrophosphate indicates the identity of the incorporated deoxynucleotide or 3′
-protected 2′
deoxynucleotide and, therefore, the identity of a base complementary thereto.
- -protected 2′
Specification