NUCLEIC ACID CLASSIFICATION

• US 20190316186A1
• Filed: 05/01/2019
• Published: 10/17/2019
• Est. Priority Date: 08/17/2012
• Status: Active Grant

• Current Assignee: Bio-NEMS Corporation
• Original Assignee: Bio-NEMS Corporation
• Inventors: Medin, David Lawrence, Lehrman, Sherwood Russ
• Granted Patent: US 10,597,704 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

A method and system for classifying a target nucleic acid includes exposing, in a test system, one or more capture probes to the target nucleic acid. The one or more capture probes is attached to a surface. A first hybridization condition is established in the test system. A first degree of hybridization of the one or more capture probes with the target nucleic acid under the first hybridization condition is determined. A second hybridization condition in the test system is established. A second degree of hybridization of the one or more capture probes with the target nucleic acid under the second hybridization condition is determined and the target nucleic acid is classified by comparing the first and the second degrees of hybridization.

Claims

1. A method for classifying a target nucleic acid, the method comprising:

exposing, in a test system, a plurality of capture probes to the target nucleic acid, the test system comprising a conditioning system, a hybridization surface, and a processor, the conditioning system being configured to apply an adjustable conditioning parameter at the plurality of capture probes, the plurality of capture probes being attached to the hybridization surface and at least two of the plurality of capture probes having different abilities to hybridize to the target nucleic acid;

establishing a first hybridization condition by adjusting the adjustable conditioning parameter in the conditioning system to a first value;

determining, by the processor, a first degree of hybridization of the plurality of capture probes with the target nucleic acid under the first hybridization condition, the first degree of hybridization comprising at least one of a first association rate and a first dissociation rate of the target nucleic acid from each capture probe under the first hybridization condition;

establishing a second hybridization condition by adjusting the adjustable conditioning parameter in the conditioning system to a second value;

determining, by the processor, a second degree of hybridization of the plurality of capture probes with the target nucleic acid under the second hybridization condition the second degree of hybridization comprising at least one of a second association rate and a second dissociation rate of the target nucleic acid from each capture probe under the second hybridization condition; and

classifying, by the processor, the target nucleic acid by determining differences between the first degree of hybridization and the second degree of hybridization with respect to each of the plurality of capture probes and comparing the differences to a model of differences of degrees of hybridization of known nucleic acids.

2. The method of claim 1, wherein:

the test system comprises one or more sensors on the surface configured to measure hybridization of the plurality of capture probes with the target nucleic acid.

3. The method of claim 2, wherein:

the one or more sensors is a resonator array configured to measure a mass of an object on at least one surface of the resonator array;

the plurality of captures probes are attached to the surface of the resonator array; and

determining the first and second degrees of hybridization of the plurality of capture probes with the target nucleic acid comprises measuring the mass on the surface of the resonator array.

4. The method of claim 1, wherein:

exposing the plurality of capture probes to the target nucleic acid comprises contacting the plurality of capture probes with a solution containing the target nucleic acid;

establishing the first hybridization condition comprises adjusting a temperature of the solution to a first temperature; and

establishing the second hybridization condition comprises adjusting the temperature of the solution to a second temperature.

5. The method of claim 1, wherein the first hybridization condition is selected to minimize hybridization between a target nucleic acid and the plurality of capture probes, and wherein the second hybridization condition is selected to favor hybridization between the target nucleic acid and the plurality of capture probes.

6. The method of claim 1, wherein classifying the target nucleic acid comprises:

progressively adjusting a plurality of hybridization conditions of the test system to favor hybridization between a target nucleic acid and the plurality of capture probes;

after each adjustment, determining a degree of hybridization of the plurality of capture probes with the target nucleic acid; and

continuing to adjust the hybridization conditions until the degree of hybridization reaches a threshold degree of hybridization.

7. The method of claim 1, wherein the test system comprises one or more first capture probes at a first location and one or more second capture probes at a second location, and wherein classifying the target nucleic acid comprises:

progressively adjusting one or more hybridization conditions of the test system;

after each adjustment, determining a respective degree of hybridization for each of the first and second locations; and

determining whether the degree of hybridization at the first location reaches a threshold degree of hybridization prior to or after the degree of hybridization at the second location reaches the threshold degree of hybridization.

8. The method of claim 1, further comprising calibrating the test system prior to exposing the plurality of capture probes to the target nucleic acid, by performing operations comprising:

attaching one or more double stranded nucleic acid capture probes to a surface of the test system;

contacting the one or more double stranded nucleic acid capture probes with a solution;

progressively adjusting one or more hybridization conditions of the test system until a degree of dissociation of the double stranded nucleic acid capture probes is reached, so that the solution contains single stranded nucleic acid and single stranded nucleic acid capture probes remain on the surface;

recording the one or more hybridization conditions at which the degree of dissociation of a first double stranded nucleic acid capture probes is reached, generating a single stranded nucleic acid; and

removing the single stranded nucleic acid from the test system.

9. The method of claim 8, further comprising calibrating the test system prior to exposing the plurality of capture probes to the target nucleic acid, by performing operations comprising:

measuring a first set of nucleic acid capture probes attached to the surface of the test system;

progressively adjusting the one or more hybridization conditions of the test system until a recorded hybridization condition is passed;

measuring a second set of nucleic acid capture probes attached to the surface of the test system; and

determining changes to the single stranded nucleic acid capture probes based on the first set of nucleic acid capture probes and the second set of nucleic acid capture probes.

43. The method of claim 1, wherein the difference indicates whether a matched or a mis-matched hybridization occurred for each of the plurality of capture probes.

10-39. -39. (canceled)

40. A method for classifying a target nucleic acid, the method comprising:

exposing, in a test system, a plurality of capture probes to the target nucleic acid, the test system comprising a conditioning system, a hybridization surface, and a processor, the conditioning system being configured to apply an adjustable conditioning parameter at the plurality of capture probes, the plurality of capture probes being attached to the hybridization surface and at least two of the plurality of capture probes having different abilities to hybridize to the target nucleic acid;

establishing a first hybridization condition by adjusting one or more conditioning parameters in the conditioning system to a first value;

determining, by the processor, a first degree of hybridization of the plurality of capture probes with the target nucleic acid under the first hybridization condition, the first degree of hybridization comprising at least one of a first association rate and a first dissociation rate of the target nucleic acid from each capture probe under the first hybridization condition;

establishing a second hybridization condition by adjusting the one or more conditioning parameters in the conditioning system to a second value;

determining, by the processor, a second degree of hybridization of the plurality of capture probes with the target nucleic acid under the second hybridization condition, the second degree of hybridization comprising at least one of a second association rate and a second dissociation rate of the target nucleic acid from each capture probe under the second hybridization condition; and

classifying, by the processor, the target nucleic acid by generating a sequence of hybridization degrees with respect to each of the plurality of capture probes based on the first degree of hybridization and the second degree of hybridization and comparing the sequence of hybridization degrees to a model sequence hybridization degree of a known nucleic acid comprising the melting temperatures for each capture probe.

41. The method of claim 40, wherein at least one of the plurality of capture probes is a perfect complementary capture probe for the known nucleic acid.

42. The method of claim 40, wherein none of capture probes is a perfect complementary capture probe for the target nucleic acid.

44. A method for classifying a target nucleic acid, the method comprising:

exposing, in a test system, a plurality of capture probes to the target nucleic acid, the test system comprising a conditioning system, a hybridization surface, and a processor, the conditioning system being configured to apply an adjustable conditioning parameter at the plurality of capture probes, the plurality of capture probes being attached to the hybridization surface and at least two of the plurality of capture probes having different abilities to hybridize to the target nucleic acid;

progressively adjusting the adjustable conditioning parameter in the conditioning system across a range of values;

for each respective capture probe, recording a respective value of the adjustable conditioning parameter at which the respective capture probe reaches a predetermined degree of hybridization with the target nucleic acid, the plurality of capture probes thus providing a sequence of conditioning parameter values comprised of the respective value for each respective capture probe; and

classifying, by the processor, the target nucleic acid as one of a plurality of test nucleic acids by comparing the sequence of conditioning parameter values to a plurality of test sequences, each test sequence comprising a sequence of conditioning values for a different test nucleic acid from the plurality of test nucleic acids.

45. The method of claim 44, wherein none of capture probes is a perfect complementary capture probe for the target nucleic acid.