Systems and methods for genetic and biological analysis
The invention relate to systems and methods for sequencing polynucleotides, as well as detecting reactions and binding events involving other biological molecules. The systems and methods may employ chamber-free devices and nanosensors to detect or characterize such reactions in high-throughput. Because the system in many embodiments is reusable, the system can be subject to more sophisticated and improved engineering, as compared to single use devices.
1. A method for sequencing a nucleic acid sample, comprising:
a. providing a plurality of particles adjacent to a sensor array, wherein an individual particle of said plurality is positioned adjacent to an individual sensor of said sensor array and is coupled to a nucleic acid molecule generated from said nucleic acid sample, and wherein said individual sensor provides a virtual wall during sequencing that isolates said individual sensor from other sensors of said sensor array;
b. hybridizing a primer to said nucleic acid molecule;
c. performing a primer extension reaction by contacting said nucleic acid molecule with nucleotide bases in the presence of a polymerase adjacent to said nucleic acid molecule;
d. detecting signals indicative of incorporation events associated with one or more of said nucleotide bases during said primer extension reaction;
e. monitoring and correcting for phase error introduced during said primer extension reaction; and
f. generating a sequence of said nucleic acid sample at reduced phase error.
2. The method of claim 1 , wherein said signals are local impedance changes accompanying said primer extension reaction.
3. The method of claim 1 , wherein said individual sensor measures a local impedance change within a Debye layer associated with said individual particle.
4. The method of claim 1 , wherein said individual sensor comprises at least two electrodes that are associated with a Debye layer of said individual particle.
5. The method of claim 1 , wherein said virtual wall is generated by a local electric field or local magnetic field.
6. The method of claim 1 , wherein said virtual wall isolates or concentrates components of said primer extension reaction.
7. The method of claim 1 , wherein phase error is corrected by (i) adding a combination of three nucleotide bases, (ii) reversibly incorporating into an in-phase polynucleotide strand a chain terminating nucleotide base, or (iii) adding an oligonucleotide clamp that hybridizes to said nucleic acid molecule and halts said primer extension reaction.
8. The method of claim 7 , further comprising denaturing, destabilizing, or degrading said clamp to continue said primer extension reaction.
9. The method of claim 8 , wherein said clamp has a 3′ terminating nucleotide base that is not extended, and wherein said 3′ terminating nucleotide base is optionally removed, thereby becoming a primer for a subsequent downstream primer extension reaction.
10. The method of claim 1 , wherein said phase error is corrected by selecting one or more nucleotide bases for incorporation to re-phase a lag by one or two bases.
11. The method of claim 1 , further comprising monitoring said signals for loss of signal that is indicative of phase error, and correcting by re-phasing to restore said signals.
12. The method of claim 1 , further comprising stockpiling said polymerase on or near said nucleic acid molecule.
13. The method of claim 12 , further comprising binding a repair protein or single stranded binding protein to said nucleic acid molecule.
14. The method of claim 1 , wherein said nucleic acid molecule is circularized.
15. The method of claim 14 , wherein said polymerase is a strand displacing polymerase.
16. The method of claim 1 , further comprising anticipating phase error based on a reference sequence.
17. A method for sequencing a nucleic acid sample at reduced phase error, comprising:
a. providing a plurality of particles adjacent to a sensor array, wherein an individual particle of said plurality is positioned adjacent to an individual sensor of said sensor array and is coupled to a nucleic acid molecule generated from said nucleic acid sample;
b. hybridizing a primer to said nucleic acid molecule;
c. performing a primer extension reaction by contacting said nucleic acid molecule with nucleotide bases in the presence of a polymerase adjacent to said nucleic acid molecule;
d. using said individual sensor, measuring local impedance changes within a Debye layer associated with said individual particle during said primer extension reaction;
e. monitoring and correcting for phase error introduced during said primer extension reaction; and
f. generating a sequence of said nucleic acid sample at reduced phase error.
18. The method of claim 17 , wherein said individual sensor comprises at least two electrodes that are coupled to a Debye layer of (i) said individual particle or (ii) nucleic acid molecules coupled to said individual particle.
19. The method of claim 17 , wherein said individual sensor provides a virtual wall during sequencing that isolates said individual sensor from other sensors of said sensor array.
20. The method of claim 17 , further comprising monitoring signals associated with said local impedance changes for loss of signal that is indicative of phase error, and correcting said phase error by re-phasing to restore said signals.
21. The method of claim 17 , wherein phase error is corrected by (i) adding a combination of three nucleotide bases, (ii) reversibly incorporating into an in-phase polynucleotide strand a chain terminating nucleotide base, or (iii) adding an oligonucleotide clamp that hybridizes to said nucleic acid molecule and halts said primer extension reaction.
22. The method of claim 17 , wherein phase error is corrected by selecting one or more nucleotide bases for incorporation to re-phase a lag by one or two bases.
23. The method of claim 17 , further comprising anticipating phase error based on a reference sequence.