SYSTEM AND METHOD FOR HIGH RESOLUTION ANALYSIS OF NUCLEIC ACIDS TO DETECT SEQUENCE VARIATIONS

• US 20110143357A1
• Filed: 11/22/2010
• Published: 06/16/2011
• Est. Priority Date: 03/28/2007
• Status: Active Grant

• Current Assignee: Xcr Diagnostics, Inc.
• Original Assignee: Signal Diagnostics, LLC
• Inventors: Caplin, Brian Erich
• Granted Patent: US 9,139,882 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Provided herein are methods for assaying a biological sample for microorganisms having drug resistant and/or drug sensitive phenotypes, wherein the methods are capable of detecting resistant and sensitive phenotypes associated with known and/or unknown mutations. In some aspects, methods are provided for detecting drug resistant Myobacterium tuberculosis (MTb), including multi-drug resistant MTb, wherein drug resistance is associated with one or more novel mutations. Also provided are systems, kits, and compositions related to such methods.

Claims

1. A method of detecting sequence variation in a target nucleic acid, comprising:

obtaining a sample comprising one or more segments of DNA;

amplifying a segment of DNA using oligonucleotide primers within a narrow temperature range, the segment of DNA including at least one polymorphism; and

assaying the amplified DNA segment for sequence variations relative to a reference sequence.

2. The method of claim 1, wherein the narrow temperature range includes the melting temperatures of the oligonucleotide primers and the melting temperature of the segment of DNA.

3. The method of claim 1, wherein the narrow temperature range comprises a range of temperatures within 2.5°

C., 5°

C., 10°

C., or 15°

C. of the melting temperature of the segment of DNA.

4. The method of claim 1, wherein the narrow temperature range is between 75°

C. and 90°

C.

5. The method of claim 1, wherein the narrow temperature range is 82°

C. to 89°

C.

6. The method of claim 1, wherein the melting temperatures of the oligonucleotide primers overlaps with the melting temperature of the segment of DNA.

7. The method of claim 1, wherein the melting temperatures of one or both of the oligonucleotide primers is the same as the melting temperature of the segment of DNA.

8. The method of claim 1, wherein the melting temperatures of the oligonucleotide primers is within 2.5°

C., 5°

C., 10°

C., or 15°

C. of the melting temperature of the segment of DNA.

9. The method of claim 1, wherein amplifying comprises annealing and denaturing of the oligonucleotide primers to the segment of DNA within the narrow temperature range.

10. The method of claim 9, wherein annealing and denaturing of the oligonucleotide primers to the segment of DNA occur in a dynamic flux.

11. The method of claim 1, wherein amplifying produces specific amplification products of the segment of DNA.

12. The method of claim 1, wherein amplifying decreases the formation of non-specific amplification products compared with amplifying within a broad temperature range.

13. The method of claim 1, wherein the amplified DNA segment is detected by a colorimetric detection readout.

14. The method of claim 13, wherein the readout is a positive or negative signal.

15. The method of claim 1, wherein the amplified DNA segment is detected by fluorescence emitted from a DNA-binding fluorescent dye.

16. The method of claim 1, wherein the assaying is conducted by high resolution melting curve analysis.

17. The method of claim 16, wherein the melting curve analysis detects one or more base pair matches between the amplified DNA segment and the reference sequence.

18. The method of claim 1, wherein the one or more segments of DNA are from an infectious microorganism and wherein the at least one polymorphism is associated with responsiveness of the microorganism to a drug.

19. The method of claim 18, wherein the reference sequence includes at least one polymorphism associated with responsiveness to the drug.

20. The method of claim 18, wherein the at least one polymorphism is associated with sensitivity to the drug.

21. The method of claim 20, wherein a variation in one or more of the amplified DNA segments indicates sensitivity to the drug.

22. The method of claim 20, wherein two or more variations in an amplified DNA segment indicates sensitivity to the drug.

23. The method of claim 18, wherein the at least one polymorphism is associated with resistance to the drug.

24. The method of claim 23, wherein a variation in one or more of the amplified DNA segments indicates resistance to the drug.

25. The method of claim 23, wherein two or more variations in an amplified DNA segment indicates resistance to the drug.