Nucleic acid sequencing by Raman monitoring of uptake of nucleotides during molecular replication
First Claim
1. A method to detect a nucleotide or nucleoside, comprising:
- a) separating a purine or pyrimidine base from a ribose or deoxyribose moiety of the nucleotide or nucleoside;
b) depositing the separated purine base or pyrimidine base on a surface enhanced Raman spectroscopy (SERS) substrate; and
c) detecting the separated purine or pyrimidine base using SERS.
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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) or surface enhanced coherent anti-Stokes Raman spectroscopy (SECARS). 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. Methods for enhancing the SERS signal of a nucleotide or nucleoside by cleaving the base from a sugar moiety are provided. Furthermore, methods for detecting single base repeats are provided.
59 Citations
44 Claims
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1. A method to detect a nucleotide or nucleoside, comprising:
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a) separating a purine or pyrimidine base from a ribose or deoxyribose moiety of the nucleotide or nucleoside;
b) depositing the separated purine base or pyrimidine base on a surface enhanced Raman spectroscopy (SERS) substrate; and
c) detecting the separated purine or pyrimidine base using SERS. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method to detect a target molecule comprising a purine base or a pyrimidine base, comprising:
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a) isolating the target molecule;
b) depositing the target molecule on a surface enhanced Raman spectroscopy (SERS) substrate;
c) detecting Raman scattering from the irradiated target molecule using surface enhanced coherent anti-Stokes Raman spectroscopy (SECARS), thereby detecting the target molecule. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23)
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24. A method to detect identical nucleotides at consecutive target positions in a template nucleic acid molecule, comprising:
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a) contacting a known number of copies of the template nucleic acid molecule with a reaction mixture comprising a primer, a polymerase, and a known initial concentration of a first nucleotide to form a post-reaction mixture, the primer or the template nucleic acid being immobilized on a surface of the reaction chamber, wherein the 3′
terminus of the primer binds to the template nucleic acid molecule upstream of a 5′
nucleotide of the consecutive target positions;
b) depositing the post-reaction mixture on a surface enhanced Raman spectroscopy (SERS) substrate;
c) detecting the first nucleotide using SERS; and
d) determining whether more than one first nucleotide was added to the consecutive target positions. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33)
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34. A method to determine a nucleotide occurrence at a target position of a template nucleic acid molecule, comprising:
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a) contacting a detectable number of template nucleic acids with a reaction mixture in a reaction chamber, the reaction mixture comprising a primer, a polymerase, and an initial concentration of a first nucleotide triphosphate, the primer or the template nucleic acid being immobilized on a surface of the reaction chamber;
b) incubating the reaction mixture to allow binding of the primer to the template nucleic acid and formation of a post-reaction mixture;
c) depositing the post reaction mixture, or a component thereof, on a surface enhanced Raman spectroscopy (SERS) substrate; and
d) detecting a Raman signal from the first nucleotide using SERS, wherein a decrease in intensity of the Raman signal of the first nucleotide in the post-reaction mixture identifies an extension reaction product, thereby identifying the nucleotide occurrence at the target position. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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Specification