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. preparing a single template nucleic acid molecule;
b. inserting the template nucleic acid molecule into a reaction chamber;
c. synthesizing a complementary nucleic acid molecule from nucleotide precursors with a synthetic reagent; and
d. monitoring the order of incorporation of nucleotide precursors into the complementary nucleic acid molecule by fluorescence resonance energy transfer (FRET) spectroscopy, wherein one or more donor tag molecules are attached to the template nucleic acid molecule and distinguishable acceptor tag molecules are attached to each type of nucleotide precursor.
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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.
60 Citations
3 Claims
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1. A method of sequencing nucleic acid molecules comprising:
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a. preparing a single template nucleic acid molecule; b. inserting the template nucleic acid molecule into a reaction chamber; c. synthesizing a complementary nucleic acid molecule from nucleotide precursors with a synthetic reagent; and d. monitoring the order of incorporation of nucleotide precursors into the complementary nucleic acid molecule by fluorescence resonance energy transfer (FRET) spectroscopy, wherein one or more donor tag molecules are attached to the template nucleic acid molecule and distinguishable acceptor tag molecules are attached to each type of nucleotide precursor.
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2. A method of sequencing nucleic acid molecules by fluorescence resonance energy transfer (FRET) comprising:
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a. attaching one or more donor tag molecules to a template nucleic acid molecule; b. attaching distinguishable acceptor tag molecules to each type of nucleotide precursor; c. reacting the template nucleic acid molecule with one or more of the nucleotide precursors in a reaction chamber, wherein an uptake of one or more of the nucleotide precursor results an energy transfer between one or more of the donor tag molecule and one or more of the acceptor tag molecule, and wherein the template nucleic acid molecule is manipulated by an optical tweezer inside the reaction chamber; d. detecting the emission from the reaction chamber; and e. determining the sequence of the template nucleic acid molecule by monitoring the emission from the reaction chamber. - View Dependent Claims (3)
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Specification