Methods to increase nucleotide signals by Raman scattering
First Claim
1. A method comprising:
- a) using an exonuclease to release unlabeled nucleotides from one end of one or more nucleic acid molecules;
b) separating the nucleotides from the exonuclease and the one or more nucleic acid molecules;
c) identifying the unlabeled nucleotides by Raman spectroscopy; and
d) determining the sequence of the nucleic acid from the identified nucleotides.
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Abstract
The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, nucleotides are covalently attached to Raman labels before incorporation into a nucleic acid. In other embodiments, unlabeled nucleic acids are used. Exonuclease treatment of the nucleic acid results in the release of labeled or unlabeled nucleotides that are detected by Raman spectroscopy. In alternative embodiments of the invention, nucleotides released from a nucleic acid by exonuclease treatment are covalently cross-linked to nanoparticles and detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Other embodiments of the invention concern apparatus for nucleic acid sequencing.
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Citations
30 Claims
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1. A method comprising:
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a) using an exonuclease to release unlabeled nucleotides from one end of one or more nucleic acid molecules;
b) separating the nucleotides from the exonuclease and the one or more nucleic acid molecules;
c) identifying the unlabeled nucleotides by Raman spectroscopy; and
d) determining the sequence of the nucleic acid from the identified nucleotides. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method comprising:
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a) sequentially removing nucleotides from one end of one or more nucleic acid molecules;
b) attaching each nucleotide to at least one nanoparticle;
d) identifying the nucleotides by Raman spectroscopy; and
e) determining the sequence of the nucleic acid. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method comprising:
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a) obtaining nucleotides that are attached to Raman labels;
b) synthesizing a nucleic acid comprising labeled nucleotides;
c) removing nucleotides from one end of the nucleic acid;
d) identifying the nucleotides by Raman spectroscopy; and
e) determining the sequence of the nucleic acid. - View Dependent Claims (23, 24, 25, 26)
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27. An apparatus comprising:
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a) a reaction chamber;
b) a microfluidic channel in fluid communication with the reaction chamber;
c) a flow-through cell in fluid communication with the microfluidic channel; and
d) a Raman detection unit operably coupled to the flow-through cell. - View Dependent Claims (28, 29, 30)
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Specification