US5945312A
Expired / Lapsed
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Synthesis of fluorophore-labeled DNA
Assignee: University of Alberta
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Filing: 1997-11-07
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Granted: 1999-08-31
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Expires: 2017-11-07
Abstract
Abstract Sequencing methods and methods for synthesizing DNA probes using mutant bacteriophage T4 DNA polymerases which have increased ability to incorporate modified nucleotides for the synthesis of long or short chains of complementary, modified, e.g., fluorophore-labeled DNA. In general, the mutant T4 DNA polymerases retain 3'->5' exonuclease activity; hence, reduction or elimination of 3'->5' exonuclease activity is not a prerequisite for efficient synthesis of a complementary fluorophore-labeled or other modified DNA. In fact, retention of 3'->5' exonuclease activity increases accuracy of DNA replication, because these exonucleases proofread or edit the product of DNA replication.
Inventors
- 👤 Myron F. Goodman
- 👤 Linda J. Reha-Krantz
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 06:07
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6848; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252; C12Q1/686; C12Q1/6869; C12Q1/6874; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333; Y10T436/24
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
43 words
A method for synthesizing a complementary DNA, from a DNA template, the improvement comprising: using a mutant Family B DNA polymerase, with increased intrinsic processivity relative to native DNA polymerase, to synthesize a complementary DNA having one or more fluorophore-labeled nucleotides incorporated therein.
Claim 2
Independent Claim
43 words
A method for synthesizing a complementary DNA, from a DNA template, the improvement comprising: using a mutant Family B DNA polymerase, with increased intrinsic processivity relative to native DNA polymerase, to synthesize a complementary DNA having one or more fluorophore-labeled nucleotides incorporated therein.
Claim 3
Dependent Claim
56 words
The method of claim 1, wherein the mutant Family B polymerase is a mutant T4 DNA polymerase selected from the group consisting of L412M-DNA polymerase, Q380K-DNA polymerase, E395K-DNA polymerase, E743K-DNA polymerase, M725I-DNA polymerase, M725V-DNA polymerase, S756P-DNA polymerase, L771F-DNA polymerase, L771H-DNA polymerase, -DNA polymerase, -DNA polymerase, V355A-DNA polymerase, E395K+L412M-DNA polymerase, L412M+E473K-DNA polymerase, E395K+L412M+E743K-DNA polymerase, and Q380K+L412M+E743K-DNA polymerase.
Claim 4
Dependent Claim
41 words
The method of claim 1, wherein the mutant Family B polymerase is a multiple mutant T4 DNA polymerase with one or more amino acid substitutions selected from the group consisting of: Q380K, E395K, E743K, M725I, M725V, S756P, L771F, L771H, and V355A.
Claim 5
Dependent Claim
26 words
The method of claim 1, further comprising the step of employing the resulting complementary DNA in single molecule sequencing, making DNA probes, or mass spectrometry sequencing.
Claim 6
Dependent Claim
20 words
The method of claim 1, wherein the mutant DNA polymerase incorporates at least two different fluorophore-labeled nucleotides into complementary DNA.
Claim 7
Dependent Claim
18 words
The method of claim 1 wherein one or more unmodified nucleotides are replaced by the corresponding fluorophore-labeled nucleotides.
Claim 8
Dependent Claim
23 words
The method of claim 1, wherein the one or more fluorophore-labeled nucleotides are selected from the group consisting of fluorescein- and rhodamine-labeled nucleotides.
Claim 9
Dependent Claim
26 words
The method of claim 1 wherein the mutant Family B DNA polymerase is selected from the group consisting of T2, T4, and T6 mutant DNA polymerases.
Claim 10
Dependent Claim
22 words
The method of claim 1 wherein the mutant Family B DNA polymerase has an amino acid substitution in highly conserved Motif A.
Claim 11
Dependent Claim
95 words
The method of claim 1, wherein the polymerase is a mixture comprising a first polymerase and a second polymerase, wherein: a. the first polymerase is a mutant T2, T4, or T6 DNA polymerase selected from the group consisting of L412M-DNA polymerase, Q380K-DNA polymerase, E395K-DNA polymerase, E743K-DNA polymerase, M725I-DNA polymerase, M725V-DNA polymerase, S756P-DNA polymerase, L771F DNA polymerase, L771H-DNA polymerase, -DNA polymerase, -DNA polymerase, V355A-DNA polymerase, E395K+L412M-DNA polymerase, L412M+E473K-DNA polymerase, E395K+L412M+E743K-DNA polymerase, and Q380K+L412M+E743K-DNA polymerase; and b. the second polymerase is selected from the group consisting of an exonuclease deficient DNA polymerase, and a thermostable DNA polymerase.
Claim 12
Dependent Claim
15 words
The method of claim 1, wherein the DNA template is primed with a first oligonucleotide.
Claim 13
Dependent Claim
18 words
The method of claim 11, wherein further synthesis of the complementary DNA is blocked by a second oligonucleotide.
Claim 14
Dependent Claim
17 words
The method of claim 1, wherein the complementary DNA is a full-length copy of the template DNA.
Claim 15
Dependent Claim
17 words
The method of claim 1 wherein the DNA template is covalently joined to a self-annealing hairpin DNA.
Claim 16
Dependent Claim
16 words
The method of claim 14, wherein the hairpin DNA contains at least one nonhydrolyzable internucleotide linkage.
Claim 17
Dependent Claim
13 words
The method of claim 15, wherein the internucleotide linkage is a phosphorothioate linkage.
Claim 18
Dependent Claim
21 words
The method of claim 14, wherein the hairpin DNA further comprises means for immobilizing a DNA molecule in a flow cell.
Claim 19
Dependent Claim
15 words
The method of claim 17, wherein the hairpin DNA contains one or more biotin residues.
Claim 20
Dependent Claim
25 words
The method of claim 1, wherein synthesizing the complementary DNA gives a duplex DNA in which one strand of the duplex DNA contains fluorophore-labeled DNA.
Claim 21
Dependent Claim
17 words
The method of claim 19, wherein the DNA template is covalently joined to a self-annealing hairpin DNA.
Claim 22
Dependent Claim
20 words
The method of claim 20, wherein the hairpin DNA further comprises means to anchor duplex DNA to a streptavidin-coated bead.
Claim 23
Dependent Claim
23 words
The method of claim 1, further comprising immobilizing the complementary DNA in a flow cell and subjecting the complementary DNA to exonuclease digestion.
Claim 24
Dependent Claim
16 words
The method of claim 22, further comprising detecting the fluorophore-labeled nucleotides released by the exonuclease digestion.
References Cited (Backward Citations)
US4711955A
Cited
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