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:
(i) contacting the target polynucleotide with a polymerase enzyme and one or amore nucleotides selected from the group consisting of A, T (U), G, and C under conditions suitable for a polymerase reaction to proceed, wherein the contact occurs in a flow cell comprising one or more immobilized polynucleotides;
(ii) observing the interaction of the polymerase enzyme with the target polynucleotide in the presence of the one or more nucleotides; and
(iii) measuring the time taken for the polymerase to bind to, and subsequently dissociate from, the target polynucleotide to identify whether the polymerase has incorporated a nucleotide onto a nascent polynucleotide paired with the target polynucleotide, and thereby identify the sequence of the target polynucleotide.
2. The method of claim 1 , wherein the one or more immobilized polynucleotides comprise(s) a primer capable of hybridization with the target polynucleotide.
3. The method of claim 1 , wherein the one or more immobilized polynucleotides comprise(s) the target polynucleotide.
4. The method of claim 1 , wherein the nascent polynucleotide comprises a primer.
5. The method of claim 1 , further comprising repeating steps (i) through (iii) with additional nucleotides.
6. The method of claim 5 , wherein steps (i) through (iii) are carried out with each of the different nucleotides in turn, until incorporation is detected.
7. The method of claim 1 , wherein a plurality of target polynucleotides are immobilized on a support material.
8. The method of claim 1 , wherein step (ii), and optionally step (iii), is carried out by measuring applied radiation.
9. The method of claim 1 , wherein step (ii), and optionally step (iii), is carried out by measuring raman scattering.
10. The method of claim 1 , wherein step (ii), and optionally step (iii), is carried out by applying a surface electromagnetic wave.
11. The method of claim 10 , wherein the surface electromagnetic wave is a surface plasmon wave.
12. The method of claim 1 , wherein step (ii), and optionally step (iii), is carried out by utilizing a non-linear imaging system.
13. The method of claim 12 , wherein the non-linear imaging system relies on second or third harmonic generation imaging.
14. The method of claim 1 , wherein the polymerase comprises a detectable label attached thereto.
15. The method of claim 14 , wherein the label is a fluorophore.
16. The method claim 14 , wherein the polymerase further comprises an energy donor label or an energy acceptor label, and wherein step (ii), and optionally step (iii), is carried out by measuring energy transfer between the fluorophore and the energy donor or acceptor.
17. The method of claim 7 , wherein each of the plurality of target polynucleotides is immobilized in an individual flow cell.
18. The method of claim 17 , wherein each of the flow cells is observed and measured individually.
19. The method of claim 1 , wherein the one or more nucleotides comprise a removable blocking group.
20. The method of claim 19 , wherein the blocking group is removed prior to, or simultaneous with, step (ii).
21. The method of claim 19 , wherein the blocking group is removed via the application of electromagnetic radiation.
22. The method of claim 1 , wherein steps (ii) and (iii) are conducted simultaneously or in sequence.
23. The method of claim 1 , further comprising determining whether a mutation in the target polynucleotide exists by comparing the sequence identification of step (iii) with the native sequence of the target polynucleotide.