High speed parallel molecular nucleic acid sequencing
First Claim
1. A method for high speed parallel molecular nucleic acid sequencing, comprising:
- (a) attaching a nucleic acid molecule to be sequenced to a substrate;
(b) adding an oligonucleotide primer under conditions where the primer anneals to the nucleic acid molecule;
(c) adding a green fluorescent protein (GFP)-polymerase and nucleotides, wherein the nucleotides are labeled with a fluorophore, and wherein each type of nucleotide comprises a different fluorophore that emits a unique emission signal corresponding to addition of that type of nucleotide as the nucleotide is incorporated into a complementary nucleic acid molecule by the GFP-polymerase;
(d) adding four deoxyribonucleoside monophosphates (dNMPs) or deoxyribonucleoside diphosphates (dNDPs), wherein the dNMPs or dNDPs comprise a fluorophore distinct from the fluorophores on the nucleotides; and
(e) detecting the unique emission signal.
1 Assignment
0 Petitions
Accused Products
Abstract
A method and device is disclosed for sequencing of nucleic acid molecules. A nucleic acid molecule is exposed to a polymerase in the presence of nucleotides. The polymerase carries a donor fluorophore, and each type of nucleotide carries a distinguishable acceptor fluorophore characteristic of the particular type of nucleotide. As the polymerase incorporates individual nucleic acid molecules into a complementary strand, a laser continuously irradiates the donor fluorophore, at a wavelength that causes it to emit an emission signal. The emission signal from the polymerase can stimulate any of the donor fluorophores (but not acceptor fluorophores), so that as a nucleotide is added, the acceptor fluorophore emits a signal associated with the type of nucleotide added to the complementary strand. The series of emission signals from the acceptor fluorophores is detected, and correlated with a sequence of nucleotides that correspond to the sequence of emission signals.
60 Citations
19 Claims
-
1. A method for high speed parallel molecular nucleic acid sequencing, comprising:
-
(a) attaching a nucleic acid molecule to be sequenced to a substrate; (b) adding an oligonucleotide primer under conditions where the primer anneals to the nucleic acid molecule; (c) adding a green fluorescent protein (GFP)-polymerase and nucleotides, wherein the nucleotides are labeled with a fluorophore, and wherein each type of nucleotide comprises a different fluorophore that emits a unique emission signal corresponding to addition of that type of nucleotide as the nucleotide is incorporated into a complementary nucleic acid molecule by the GFP-polymerase; (d) adding four deoxyribonucleoside monophosphates (dNMPs) or deoxyribonucleoside diphosphates (dNDPs), wherein the dNMPs or dNDPs comprise a fluorophore distinct from the fluorophores on the nucleotides; and (e) detecting the unique emission signal. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
-
-
16. A method for high speed parallel molecular nucleic acid sequencing, comprising:
-
(a) attaching a green fluorescent protein (GFP)-polymerase to a substrate; (b) adding a nucleic acid molecule to be sequenced; (c) adding a oligonucleotide primer under conditions where the primer anneals to the nucleic acid molecule to be sequenced; (d) adding nucleotides, wherein the nucleotides are labeled with a fluorophore, and wherein each type of nucleotide comprises a different fluorophore that emits a unique emission signal corresponding to addition of that type of nucleotide as the nucleotide is incorporated into a complementary nucleic acid molecule by the GFP-polymerase; (e) adding four deoxyribonucleoside monophosphates (dNMPs) or deoxyribonucleoside diphosphates (dNDPs), wherein the dNMPs or dNDPs comprise a fluorophore distinct from the fluorophores on the nucleotides; and (f) detecting the unique emission signal. - View Dependent Claims (17, 18, 19)
-
Specification