Sequence Determination By Use Of Opposing Forces

• US 20110059864A1
• Filed: 09/07/2010
• Published: 03/10/2011
• Est. Priority Date: 09/07/2009
• Status: Abandoned Application

• Current Assignee: Caerus Molecular Diagnostics Incorporated
• Original Assignee: Caerus Molecular Diagnostics Incorporated
• Inventors: Chow, Andrea, Parce, John Wallace, Farinas, Javier
• Application Number:
  US12/877,103
• Publication Number:
  US 20110059864A1
• Time in Patent Office: Days
• US Class Current: 506/12
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

The present teachings relate to systems, methods, and the like, for analyzing biological polymers, by use of opposing forces. Among other things, the present teachings can be used to determine sequence information, such as in genetic sequencing and genotyping applications. Various embodiments are described for efficient, high throughput sequencing of nucleic-acid molecules, such as DNA. Various embodiments are described wherein nucleic-acid sequence information is determined without the need or use of extrinsic labels. As well various embodiments of methods, systems, and the like, are described, which can provide long and accurate read lengths for low-cost nucleic acid sequencing.

Claims

1. A system for determining sequence information of a polymeric analyte, wherein the analyte includes (i) at least one nucleic-acid molecule comprising a number of monomer units and (ii) a quantity of charge, with the quantity being a function of the number;

said system comprising;

first and second sources of force, each configured to apply, when active, a respective force that acts upon said analyte; and

wherein the applied forces include vector components in opposing relation; and

further wherein the forces include an electrical force, strongly dependent on the quantity of charge of said analyte, and at least one additional force, weakly or non-dependent on the quantity of charge of said analyte;

a reagent region, and a composition of matter therein;

wherein said composition of matter comprises a mixture being characterized by an enzyme activity effective to change the number of monomer units, when in effective proximity with the analyte;

a support element comprising a microbead, characterized by a detectable parameter, disposed in close association with the analyte such that a change in the number of monomer units, accompanied by a change in the charge, effects a change in the detectable parameter;

a surface and a tether, with the tether comprising a biological polymer;

wherein the tether extends between the surface and the microbead in a manner to tether the microbead to said surface; and

wherein said tether can provide a spring-like, restoring force;

a pathway leading the composition into effective proximity with the analyte;

a detector configured to detect a change in the detectable parameter of the support element; and

,

a processing unit, adapted to receive signal information from the detector, and programmed to process the information and provide sequence information relating to one or more monomer units of the polymeric analyte.

2-6. -6. (canceled)

7. A method for analyzing sequence information of a polymeric analyte, with said analyte including (a) at least one nucleic-acid molecule comprising a DNA molecule, and (b) a number of monomer units and a net charge which is a function of the number;

the method comprising;

(i) supporting the analyte with a particle;

wherein the particle includes a detectable parameter that is a function of the charge;

(ii) forming an array comprising the analyte-supporting particle and a plurality of additional analyte-supporting particles;

(iii) tethering the particles of the array to a surface with respective tethers which, at least in part, can provide a restoring force;

(iv) applying opposing forces so as to act upon the array of analyte-supporting particles;

wherein one of the forces is characterized by a dependence on the charge of the analyte;

(v) detecting and measuring the parameter;

(vi) changing the number of monomer units of the analyte;

wherein said changing is carried out by synthesizing nucleic acid, cleaving nucleic acid, binding an oligonucleotide, displacing a bound oligonucleotide, or ligating nucleic acids;

(vii) detecting and measuring for a consequential change in the parameter; and

(viii) analyzing any changed parameter, thereby determining sequence information relating to the analyte;

wherein a plurality of the steps of said method are carried out substantially simultaneously on said array of particles; and

further wherein a plurality of contiguous nucleic acids of DNA are sequenced so as to provide single-base resolution sequencing.

8-20. -20. (canceled)

21. A method for sequencing one or more nucleic-acid polymers, comprising:

(i) generating a plurality of analyte complexes, with individual complexes of the plurality including (a) a bead support, at least one nucleic-acid polymer, and a quantity of associated nucleic acids, wherein individual nucleic acids of the quantity are unlabeled and include a charge, (b) a net charge that depends on the quantity of associated nucleic acids, and (c) a kinematic property that depends on the net charge;

(ii) forming an analyte array comprising the plurality of analyte complexes;

(iii) acting upon the analyte array with a plurality of forces, with the forces including (a) an electrical force and a restoring force, and (b) vector components disposed in opposing directions;

(iv) imaging the analyte array to detect for signals corresponding to the kinematic property, to obtain a first set of measurements;

(v) subjecting the analyte array to reagents and conditions suitable for a sequencing-by-synthesis reaction, whereby the quantity of associated nucleic acids of one or more analyte complexes of the analyte array can be changed in a sequence-dependent manner;

(vi) imaging the analyte array to detect for signals corresponding to the kinematic property, to obtain a second set of measurements;

(vii) comparing first and second sets of measurements to identify differences indicating one or more sequencing-by-synthesis incorporation events; and

(viii) repeating steps (iii) through (vii);

whereby at least one or more portions of said at least one nucleic-acid polymer of the analyte complexes are sequenced.

22. The method of claim 21, wherein said at least one nucleic-acid polymer is DNA or RNA;

wherein said imaging steps are effected at least in part using an imaging detector; and

further wherein said restoring force is provided at least in part by a tether or a dielectrophoretic trap.

23. The method of claim 21, wherein the plurality of forces, recited in step (iii), act upon the analyte array at least during both of the imaging steps, recited in steps (iv) and (vi).

24. A system for sequencing nucleic-acid polymers, comprising:

means providing a plurality of analysis-sites for receiving a plurality of complexes for analysis in parallel, with each complex including, in combination, one or more nucleic-acid polymers, a means for supporting said one or more nucleic-acid polymers and a quantity of associated charged nucleic acids;

wherein the complex is characterized by a net charge dependent on the quantity of associated charged nucleic acids, and a kinematic property dependent on the net charge;

means for applying plural forces for simultaneously acting at the plurality of analysis sites, wherein the forces include vector components disposed in opposing directions, and at least one the forces includes a dependence on the net charge;

means capable of changing the quantity of associated charged nucleic acids by sequence-dependent addition of nucleic-acid monomers;

means for measuring the kinematic property at (i) a time before and (ii) a time at or after an action of the means capable of changing;

means for comparing measurements to determine a difference; and

means for processing results generated by the means for comparing, to thereby determine one or more sequences.

25. A system for analysis of at least one user-provided analyte complex, which complex includes a biological-polymer comprising a sequence of monomers, an associated support and a quantity of associated charged moieties;

wherein the complex includes a net charge that depends on the quantity of associated charged moieties, and a kinematic property that depends on the net charge;

the system comprising;

an analysis site adapted to receive the analyte complex;

means for applying opposing forces at the analysis site, with at least one of the forces including a strong dependence on the net charge, and another of the forces including no more than a weak dependence on the net charge;

means capable of changing the quantity of associated charged moieties, with such capability including a dependence on a given sequence;

means for detecting the kinematic property at (i) a time before and (ii) a time at or after action of the means capable of changing;

means for measuring detected kinematic properties;

means for comparing measurements of detected kinematic properties; and

means for analyzing results relating to detected kinematic properties, and determining sequence information.

26. The system of claim 25, further comprising:

means for determining a difference between measurements of detected kinematic properties; and

means for quantifying the difference in the event the determined difference is non-zero.

27. The system of claim 25, wherein a CMOS device comprises, at least in part, two or more of said means for detecting, means for measuring, means for comparing, means for analyzing, means for determining a difference, and means for quantifying.

28. The system of claim 25, wherein said means for applying opposing forces comprises (i) a source for providing an electrical force that is dependent on the net charge, and (ii) a means for providing a restoring force, which exhibits little to no dependence on the net charge.

29. A method for analyzing a sequence characteristic of at least one polymeric molecule, the method comprising:

(i) forming an analyte complex that includes (a) at least one polymeric-molecule analyte, a support therefore and a quantity of associated charged moieties, (b) a net charge that depends on the quantity of associated charged moieties, and (c) a kinematic property that depends on the net charge;

(ii) acting upon the analyte complex with plural forces, which include vector components disposed in opposing directions, with at least one of the forces being dependent on the net charge of the analyte complex;

(iii) measuring the kinematic property of the analyte complex;

(iv) next, subjecting the analyte complex to an agent or event capable of changing the quantity of charged moieties associated with the polymeric molecule, with such capability being dependent on the sequence characteristic;

(v) then, again measuring the kinematic property of the analyte complex; and

(vi) comparing measurements from steps (iii) and (v) to determine a difference, thereby obtaining information relating to the sequence characteristic.

30. The method of claim 29, further comprising:

repeating steps (ii) through (vi) one or more times, thereby obtaining additional information for one or more monomer units comprising said at least one polymeric-molecule analyte.

31. The method of claim 30, wherein said at least one polymeric-molecule analyte comprises a biological polymer;

and wherein the system further comprises the step of processing the information so as to define a sequence of contiguous monomer units.

32. The method of claim 31, wherein said method is carried out on at least 1,000 analyte complexes in parallel, with said at least 1,000 analyte complexes being simultaneously imaged.

33. The method of claim 31, wherein the biological polymer comprises a nucleic-acid polymer, and the charged moieties comprise nucleic acids.

34. The method of claim 33, wherein said analyte complex includes no more than a single nucleic-acid polymer.

35. The method of claim 33, wherein said analyte complex includes a plurality of nucleic-acid polymers, wherein said plurality of nucleic-acid polymers comprises replicates of a nucleic-acid polymer of interest.

36. The method of claim 35, wherein step (i) comprises forming a plurality of distinct, unique analyte complexes.

37. The method of claim 33, wherein said support of the analyte complex comprises a particle.

38. The method of claim 37, wherein said kinematic property is position, linear velocity, rotational velocity, acceleration, or a combination of the foregoing.

39. The method of claim 37, wherein plural analyte complexes are analyzed in parallel, with each including a particle supporting one or more associated nucleic-acid polymers;

and wherein at least 1,000 analyte complexes are analyzed in parallel; and

further wherein said at least 1,000 analyte complexes are configured in an array.

40. The method of claim 39, further wherein said measuring steps include imaging said array.

41. The method of claim 31, wherein said plural forces include an electrical force, which electrical force comprises, at least in part, said at least one of the forces that is dependent on the net charge.

42. The method of claim 41, wherein the method steps are carried out on at least 1,000 analyte complexes in parallel;

and wherein the method further comprises the step of configuring said at least 1,000 analyte complexes so as to define an array.

43. The method of claim 41, wherein said support of said analyte complex comprises a particle;

and further wherein one or more of said plural forces comprises a restoring force.

44. The method of claim 43, wherein said restoring force is a tethering force, an optical trapping force, a magnetic trapping force, a dielectrophoretic trapping force, or a combination thereof.

45. The method of claim 44, wherein said restoring force comprises a tethering force, provided at least in part by a polymeric molecule tethering said analyte complex.

46. The method of claim 33, wherein said support comprises a particle;

and wherein said nucleic-acid polymer comprises at least one polynucleotide; and

further wherein step (iv) includes changing the amount of nucleic acids associated with the at least one polynucleotide.

47. The method of claim 46, wherein said changing the amount of nucleic acids associated with the at least one polynucleotide includes providing one or more primed polynucleotides, and sequentially subjecting said one or more primed polynucleotides to reagents and conditions suitable for sequencing-by-synthesis reactions.

48. The method of claim 46, wherein said changing the amount of nucleic acids is effected by one of:

(a) synthesizing one or more nucleic acids to the polynucleotide bound to the particle, (b) binding one or more oligonucleotides to the polynucleotide bound to the particle, (c) displacing one or more bound oligonucleotides from the polynucleotide bound to the particle, or (d) ligating one or more nucleic acids to the polynucleotide bound to the particle.

49. A system for analyzing a sequence characteristic of at least one polymeric molecule;

the system for use with an analyte complex that includes (a) at least one polymeric-molecule analyte, a support therefore and a quantity of associated charged moieties, (b) a net charge that depends on the quantity of associated charged moieties, and (c) a kinematic property that depends on the net charge;

the system further for use with at least one analytic reagent mixture capable of changing the quantity of associated charged moieties, with such capability being dependent on the sequence characteristic;

the system comprising;

a reaction region, including an analysis site adapted to receive and support the analyte complex, when provided for analysis;

first and second sources of force, each configured to apply, when in an activated state, a respective force along the reaction region, with the applied forces (i) arranged to act at the analysis site, and (ii) including vector components disposed in opposing directions;

a reagent source, an entry to the reaction region, and a reagent-delivery path extending from the reagent source, through the entry, and to the analysis site;

wherein the path is configured to facilitate contact between an analytic reagent mixture, when provided by the source for delivery along the path to the analysis site, and the analyte complex;

a detector operatively configured (i) to observe at least a portion of the reaction region, including the analysis site, while the sources of force are in the activated state and the applied forces present, and (ii) to detect for first and second signals, separated by a temporal interval, relating to the kinematic property of the analyte complex;

with the temporal interval comprising a duration sufficient to allow delivery of the analytic reagent mixture to the analysis site; and

a signal processor adapted to receive detected first and second signals, and configured to compare the signals and determine a difference, thereby obtaining information relating to the sequence characteristic.

50. The system of claim 49, further comprising one or more additional sources of force configured to apply, when in an activated state, one or more respective forces along the reaction region.

51. The system of claim 50, wherein said sources of force include one or more sources configured for providing an electrical force and a restoring force.

52. The system of claim 49, further comprising a flow cell, with said flow cell including:

(i) a chamber defining said reaction region, (ii) at least one reservoir defining said reagent source, and, (iii) one or more channels between said one or more reservoirs and said chamber.

53. The system of claim 49, wherein said detector comprises an imaging array detector which includes an array of pixels.

54. The system of claim 53, wherein said detector comprises a CMOS sensor and a logic device programmed for calculating kinetic-parameter information from a plurality of sensor images.

55. The system of claim 49, further comprising a processing means programmed to interpret detector signal information and provide output corresponding to sequences of nucleic acids.