Apparatus and method for sequencing nucleic acid molecules
View Patent ↗The sequence of a target polynucleotide can be determined by: (i) contacting the target polynucleotide with a polymerase enzyme and one of the nucleotides A, T(U), G and C under conditions suitable for the polymerase reaction to proceed; (ii) measuring the time taken for the polymerase to bind to and subsequently dissociate from the target polynucleotide, to thereby determine whether the polymerase has incorporated the nucleotide onto the target polynucleotide; (iii) optionally repeating steps (i) and (ii) with additional nucleotides, to thereby identify the sequence of the target polynucleotide.
1. A method for sequencing a target polynucleotide, comprising:
a. immobilizing the target polynucleotide on a support of an apparatus for sequencing a polynucleotide, comprising:
i. a flow cell comprising a fluid flow path;
ii. a monochromatic laser; and
iii. a total internal reflection microscope,
wherein the support is positioned within the fluid flow path of the flow cell such that, upon immobilization of a target polynucleotide, the target polynucleotide is positioned in a reaction site within at least a portion of the fluid flow path; and wherein the laser is configured to emit an optical beam directed at the reaction site, and wherein a total internal reflection microscope is focused on the reaction site such that a polymerization reaction associated with the target polynucleotide can be monitored over time;
b. contacting the target polynucleotide with a polymerase enzyme and a nucleotide selected from the group consisting of A, T(U), G, and C under conditions suitable for a polymerase reaction to proceed, wherein the nucleotide comprises a removable blocking group;
c. observing the interaction of the polymerase enzyme and the nucleotide with the target polynucleotide;
d. determining whether the polymerase has incorporated the nucleotide onto the primer sequence; and
e. repeating steps (b)-(d) one or more times, thereby identifying the sequence of the target polynucleotide.
2. The method of claim 1 , wherein unbound nucleotides are removed from the reaction site prior to step (c).
3. The method of claim 1 , wherein the solid support further comprises one or more target polynucleotides immobilized thereon.
4. The method of claim 1 , wherein the solid support further comprises one or more immobilized primer sequences that are complementary to the target polynucleotide.
5. The method of claim 1 , wherein the apparatus is configured to monitor the polymerization reaction by monitoring changes in fluorescent signal intensity over time.
6. The method of claim 1 , wherein the removable blocking group comprises a 5′ blocking group.
7. The method of claim 1 , wherein the removable blocking group comprises a radiation labile blocking group.