Nucleic acid sequencing by Raman monitoring of uptake of nucleotides during molecular replication
First Claim
1. A method to detect a nucleotide, nucleoside, or base, comprising:
- a) depositing the nucleotide, nucleoside, or base on a substrate comprising aluminum or comprising a metal-coated nanostructure;
b) irradiating the deposited nucleotide, nucleoside, or base; and
c) detecting Raman spectra from the irradiated nucleotide, nucleoside, or base, thereby detecting the nucleotide, nucleoside, or base.
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Abstract
The methods and apparatus disclosed herein are useful for detecting nucleotides, nucleosides, and bases and for nucleic acid sequence determination. The methods involve detection of a nucleotide, nucleoside, or base using surface enhanced Raman spectroscopy (SERS). The detection can be part of a nucleic acid sequencing reaction to detect uptake of a deoxynucleotide triphosphate during a nucleic acid polymerization reaction, such as a nucleic acid sequencing reaction. The nucleic acid sequence of a synthesized nascent strand, and the complementary sequence of the template strand, can be determined by tracking the order of incorporation of nucleotides during the polymerization reaction.
47 Citations
35 Claims
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1. A method to detect a nucleotide, nucleoside, or base, comprising:
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a) depositing the nucleotide, nucleoside, or base on a substrate comprising aluminum or comprising a metal-coated nanostructure;
b) irradiating the deposited nucleotide, nucleoside, or base; and
c) detecting Raman spectra from the irradiated nucleotide, nucleoside, or base, thereby detecting the nucleotide, nucleoside, or base. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. An apparatus comprising:
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a) a reaction chamber containing a single template nucleic acid molecule attached to an immobilization surface;
b) a channel in fluid communication with the reaction chamber; and
c) a Raman detection unit operably coupled to the channel. - View Dependent Claims (19, 20, 21, 22, 23)
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24. A method to determine a nucleotide occurrence at a target position of one or more template nucleic acid molecules, comprising:
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a) contacting the one or more template nucleic acid molecules with a reaction mixture comprising a primer, a polymerase, and an initial concentration of a first nucleotide, wherein the 3′
nucleotide of the primer binds to the template nucleic acid adjacent to the target nucleotide position to form a post-reaction mixture; and
b) determining the concentration of the first nucleotide in the post-reaction mixture using Raman spectroscopy, wherein a decrease in the post-reaction concentration of the first nucleotide identifies an extension reaction product, thereby identifying the nucleotide occurrence at the target position; and
c) repeating steps a-b with a different nucleotide until the nucleotide occurrence is identified. - View Dependent Claims (25, 26, 27, 28)
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29. A method to sequence one or more nucleic acid molecules, comprising:
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a) contacting the one or more template nucleic acid molecules with nucleotides, a primer, and a polymerase to form a reaction mixture, the one or more template nucleic acid molecules or the primer being immobilized on a solid support;
b) synthesizing one or more complementary strands to the one or more template nucleic acid molecules;
d) measuring the concentrations of the nucleotides in the reaction mixture by Raman spectroscopy; and
e) determining the sequence of the template nucleic acid from the nucleotides incorporated into the complementary strand. - View Dependent Claims (30, 31, 32, 33, 34, 35)
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Specification