Nucleic acid sequencing by raman monitoring of uptake of precursors during molecular replication
First Claim
1. A method of sequencing nucleic acid molecules comprising:
- a) inserting multiple copies of a template nucleic acid molecule into a reaction chamber;
b) synthesizing a complementary nucleic acid molecule from nucleotide precursors with a polymerase;
c) monitoring the order of incorporation of nucleotide precursors into the complementary nucleic acid molecule by surface enhanced Raman scattering, surface enhanced resonance Raman scattering, stimulated Raman scattering, inverse Raman, stimulated gain Raman spectroscopy, hyper-Raman scattering or coherent anti-Stokes Raman scattering, wherein a tag molecule is attached to each nucleotide precursor; and
d) determining the sequence of the template nucleic acid molecule from the order of incorporation of nucleotide precursors into complementary nucleic acid molecules.
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Abstract
The methods, compositions and apparatus disclosed herein are of use for nucleic acid sequence determination. The methods involve isolation of one or more nucleic acid template molecules and polymerization of a nascent complementary strand of nucleic acid, using a DNA or RNA polymerase or similar synthetic reagent. As the nascent strand is extended one nucleotide at a time, the disappearance of nucleotide precursors from solution is monitored by Raman spectroscopy or FRET. The nucleic acid sequence of the nascent strand, and the complementary sequence of the template strand, may be determined by tracking the order of incorporation of nucleotide precursors during the polymerization reaction. Certain embodiments concern apparatus comprising a reaction chamber and detection unit, of use in practicing the claimed methods. The methods, compositions and apparatus are of use in sequencing very long nucleic acid templates in a single sequencing reaction.
27 Citations
13 Claims
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1. A method of sequencing nucleic acid molecules comprising:
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a) inserting multiple copies of a template nucleic acid molecule into a reaction chamber;
b) synthesizing a complementary nucleic acid molecule from nucleotide precursors with a polymerase;
c) monitoring the order of incorporation of nucleotide precursors into the complementary nucleic acid molecule by surface enhanced Raman scattering, surface enhanced resonance Raman scattering, stimulated Raman scattering, inverse Raman, stimulated gain Raman spectroscopy, hyper-Raman scattering or coherent anti-Stokes Raman scattering, wherein a tag molecule is attached to each nucleotide precursor; and
d) determining the sequence of the template nucleic acid molecule from the order of incorporation of nucleotide precursors into complementary nucleic acid molecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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Specification