Methods for determining nucleotide sequence information
First Claim
1. A method to determine a nucleotide sequence of a target nucleic acid, comprising a) contacting the nucleic acid, or a fragment thereof, with a population of capture oligonucleotide probes bound to a substrate at a series of spot locations, to form probe-target duplex nucleic acids comprising single-stranded overhangs;
- b) contacting the probe-target duplex nucleic acids with a population of Raman-active oligonucleotide probes to allow binding of the Raman probes to the single-stranded overhangs, wherein each Raman-active oligonucleotide probe generates a distinct Raman signature;
c) detecting Raman-active oligonucleotide probes that bind the template nucleic acid using Raman spectroscopy; and
e) identifying the location of the spot for each of the captured Raman-active oligonucleotide probes, thereby determining a nucleotide sequence of the target nucleic acid.
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Abstract
Provided herein, is a nucleic acid sequencing method based on detection of Raman signatures of oligonucleotide probes. Raman signatures of individually captured nucleic acid probes, optionally labeled by a Raman label or a positively charged enhancer, are detected. The sequences of captured probes are used to identify the nucleotide sequences of captured probes and complementary target nucleic acids, which are then aligned and used to obtain nucleic acid sequence information. In another embodiment, a method is provided for determining a nucleotide occurrence at a target nucleotide position of a target nucleic acid, that utilizes binding of the target nucleic acid to a labeled oligonucleotide probe that binds to the target nucleic acid, wherein the labeled oligonucleotide probe includes a first label and a second label, the first label being capable of affecting an optical property of the second label.
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Citations
36 Claims
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1. A method to determine a nucleotide sequence of a target nucleic acid, comprising
a) contacting the nucleic acid, or a fragment thereof, with a population of capture oligonucleotide probes bound to a substrate at a series of spot locations, to form probe-target duplex nucleic acids comprising single-stranded overhangs; -
b) contacting the probe-target duplex nucleic acids with a population of Raman-active oligonucleotide probes to allow binding of the Raman probes to the single-stranded overhangs, wherein each Raman-active oligonucleotide probe generates a distinct Raman signature;
c) detecting Raman-active oligonucleotide probes that bind the template nucleic acid using Raman spectroscopy; and
e) identifying the location of the spot for each of the captured Raman-active oligonucleotide probes, thereby determining a nucleotide sequence of the target nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A detection system comprising:
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a) a Raman spectrometer comprising a light source;
b) a Raman active surface in optical communication with the light source; and
c) a population of Raman-active oligonucleotide probes comprising an undetectable oligonucleotide backbone associated with a positively charged enhancer, wherein the Raman-active oligonucleotide probes are deposited on the Raman active surface. - View Dependent Claims (20, 21)
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22. A method to determine a nucleotide occurrence at a target nucleotide position of a template nucleic acid, comprising:
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a) providing a labeled oligonucleotide probe that binds to the target polynucleotide, wherein the labeled oligonucleotide probe comprises a first label and a second label, the first label affecting the Raman spectra or fluorescent signal generated by the second label based on the orientation of the first label to the second label;
b) contacting the labeled oligonucleotide probe with the target polynucleotide to form a probe-target complex; and
c) detecting the fluorescent signal or Raman spectra generated by the second label, wherein the nucleotide occurrence at the target nucleotide position affects the orientation of the first label to the second label, thereby affecting the fluorescent signal or Raman spectra generated by the second label and allowing determination of the nucleotide occurrence at the target nucleotide position. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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33. A method for detecting a nucleic acid, comprising:
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a) irradiating the nucleic acid with light, wherein the nucleic acid comprises a positively-charged enhancer; and
b) detecting a Raman signal generated by the irradiated nucleic acid. - View Dependent Claims (34, 35, 36)
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Specification