System and method for high resolution analysis of nucleic acids to detect sequence variations

• US 9,139,882 B2
• Filed: 11/22/2010
• Issued: 09/22/2015
• Est. Priority Date: 03/28/2007
• Status: Active Grant

• Current Assignee: Xcr Diagnostics, Inc.
• Original Assignee: Fluoresentric, Inc.
• Inventors: Caplin, Brian Erich
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US12/951,710
• Publication Number:
  US 20110143357A1
• Time in Patent Office: 1,765 Days
• Field of Search: None
• US Class Current: 1/1
• 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 for nucleic acid sequence amplification, comprising:

identifying a target nucleic acid sequence from one or more segments of DNA, said target nucleic acid sequence having primers with melting temperatures that are within at least 15°

C. of the melting temperature of the target nucleic acid sequence;

obtaining a pair of oligonucleotide primers with an annealing curve (T_A) that overlaps with a denaturation curve (T_D) of the target nucleic acid sequence, in such a manner as to minimize the temperature range between the higher of the melting temperature of a pair of oligonucleotide primers and the melting temperature of the target nucleic acid sequence, wherein the temperature range does not exceed 15°

C.; and

amplifying the target nucleic acid sequence by thermocycling the target nucleic acid sequence and the pair of oligonucleotide primers within a 15°

C. temperature range,

wherein thermocycling comprises;

i. denaturing the target nucleic acid sequence; and

ii. annealing of the pair of oligonucleotide primers; and

iii. extension of the target nucleic acid sequence by the pair of oligonucleotide primers,

wherein an upper thermal cycle temperature in the thermocycling is selected to minimize non target denaturation and maximize target denaturation.

2. The method of claim 1, wherein the temperature range between the melting temperature of one of the pair of oligonucleotide primers and the melting temperature of the target nucleic acid sequence is not greater than 2.5°

C., wherein the one of the pair of oligonucleotide primers has a greater melting temperature range difference with the melting temperature of the target nucleic acid sequence than the second of the pair of oligonucleotide primers.

3. The method of claim 1, wherein the temperature range between the higher of the melting temperature of a pair of oligonucleotide primers and the melting temperature of the target nucleic acid sequence is 82°

C. to 89°

C.

4. The method of claim 1, wherein the melting temperature of at least one of the oligonucleotide primers is the same as the melting temperature of the target nucleic acid sequence.

5. The method of claim 1, wherein the melting temperature of at least one of the oligonucleotide primers is within 2.5°

C. of the melting temperature of the target nucleic acid sequence.

6. The method of claim 1, wherein annealing and denaturing of the oligonucleotide primers to the target nucleic acid sequence occur in a dynamic flux.

7. The method of claim 1, wherein amplifying produces specific amplification products of the target nucleic acid sequence.

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

9. The method of claim 1, wherein the amplified target nucleic acid sequence is detected by a colorimetric detection readout.

10. The method of claim 9, wherein the readout is a positive or negative signal.

11. The method of claim 1, further comprising detecting the amplified target nucleic acid sequence by fluorescence emitted from a DNA-binding fluorescent dye.

12. The method of claim 1, further comprising assaying by high resolution melting curve analysis the amplified target nucleic acid sequence for sequence variations relative to a reference sequence.

13. The method of claim 12, wherein the melting curve analysis detects one or more base pair matches between the amplified target nucleic acid sequence and the reference sequence.

14. The method of claim 1, wherein the one or more segments of DNA are from an infectious microorganism and wherein the target nucleic acid sequence comprises at least one polymorphism that is associated with responsiveness of the microorganism to a drug.

15. The method of claim 14, further comprising identifying a reference sequence that includes at least one polymorphism associated with responsiveness to the drug.

16. The method of claim 14, wherein the at least one polymorphism is associated with sensitivity to the drug.

17. The method of claim 15, wherein the at least one polymorphism of the reference sequence indicates sensitivity to the drug.

18. The method of claim 14, wherein the at least one polymorphism is associated with resistance to the drug.

19. The method of claim 15, wherein the at least one polymorphism of the reference sequence indicates resistance to the drug.

20. The method of claim 1, wherein the target nucleic acid sequence includes at least one polymorphism.

21. The method of claim 20, further comprising assaying the amplified target nucleic acid sequence for sequence variations relative to a reference sequence.

22. The method of claim 1, wherein the thermocycling is carried out within a temperature range that is within 2 degrees of the temperature range between the melting temperature of the oligonucleotide primers and the melting temperature of the target nucleic acid sequence.

23. The method of claim 1, wherein the thermocycling is carried out within the temperature range that is between the melting temperature of the oligonucleotide primers and the melting temperature of the target nucleic acid sequence.

24. The method of claim 1, wherein the temperature range between the melting temperature of one of the oligonucleotide primers and the melting temperature of the target nucleic acid sequence is not greater than 5°

C., wherein the one of the oligonucleotide primers has the greatest melting temperature difference with the melting temperature of the target nucleic acid sequence.

25. The method of claim 1, wherein the temperature range between the melting temperature of one of the oligonucleotide primers and the melting temperature of the target nucleic acid sequence is not greater than 10°

C., wherein the one of the oligonucleotide primers has the greatest melting temperature difference with the melting temperature of the target nucleic acid sequence.

26. The method of claim 1, wherein the melting temperature of at least one of the oligonucleotide primers is within 5°

C. of the melting temperature of the target nucleic acid sequence.

27. The method of claim 1, wherein the melting temperature of at least one of the oligonucleotide primers is within 10°

C. of the melting temperature of the target nucleic acid sequence.

41. The method of claim 1, wherein each of the pair of oligonucleotide primers is present in an approximately equimolar concentration.

28. A method for rapid amplification of a target nucleic acid sequence, comprising:

identifying a target nucleic acid sequence from one or more segments of DNA;

determining the melting temperature of the target nucleic acid sequence via an iterative process comprising amplifying the target nucleic acid sequence and then determining the melting temperature of the target nucleic acid sequence via melting curve analysis; and

generating a pair of oligonucleotide primers having an annealing curve (T_A) that overlaps with a denaturation curve (T_D) of the target nucleic acid sequence, in such a manner as to minimize the temperature range between the melting temperatures (Tm) of the pair of oligonucleotide primers and the melting temperature (Tm) of the target nucleic acid sequences; and

amplifying the target nucleic acid sequence by thermocycling the target nucleic acid sequence and the pair of oligonucleotide primers within a 15°

C. temperature range,

wherein thermocycling comprises;

i. denaturing the target nucleic acid sequence; and

ii. annealing of the pair of oligonucleotide primers; and

iii. extension of the target nucleic acid sequence by the pair of oligonucleotide primers.

29. The method of claim 28, wherein the melting temperatures (Tm) of the pair of oligonucleotide primers are within 2.5°

C. of the melting temperature (Tm) of the target nucleic acid sequence.

30. The method of claim 28, wherein the melting temperatures (Tm) of the pair of oligonucleotide primers are within 5°

C. of the melting temperature (Tm) of the target nucleic acid sequence.

31. The method of claim 28, wherein the melting temperatures (Tm) of the pair of oligonucleotide primers are within 10°

C. of the melting temperature (Tm) of the target nucleic acid sequence.

32. The method of claim 28, wherein the melting temperatures (Tm) of the pair of oligonucleotide primers are within 15°

C. of the melting temperature (Tm) of the target nucleic acid sequence.

33. The method of claim 28, wherein the melting temperatures (Tm) of the pair of oligonucleotide primers and the melting temperature (Tm) of the target nucleic acid sequence are between 75°

C. and 90°

C.

34. The method of claim 28, wherein the melting temperatures (Tm) of the pair of oligonucleotide primers and the melting temperature (Tm) of the target nucleic acid sequence are between 82°

C. and 89°

C.

35. The method of claim 28, further comprising assaying a resulting amplicon for sequence variations relative to a reference sequence.

36. The method of claim 28, further comprising adjusting the pH of the amplification reaction to increase the specificity of selectively amplifying the target nucleic acid over other nucleic acids.

42. The method of claim 28, wherein each of the pair of oligonucleotide primers is present in an approximately equimolar concentration.

37. A dynamic flux method of nucleic acid sequence amplification, comprising:

a. combining a pair of forward and reverse oligonucleotide primers with a target nucleic acid sequence to be amplified; and

b. amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence within a 15°

C. temperature range that is defined by the area contained within the overlap of an annealing curve (T_A) of the pair of oligonucleotide primers and the denaturation curve (T_D) of the target nucleic acid sequence,

wherein each forward and reverse oligonucleotide primer has a melting temperature (T_m) within 15°

C. of the T_m of the target nucleic acid sequence, and

wherein thermocycling comprises;

i. denaturing the target nucleic acid sequence; and

ii. annealing of the forward and reverse oligonucleotide primers; and

iii. extension of the target nucleic acid sequence by the forward and reverse oligonucleotide primers.

38. The dynamic flux method of claim 37, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 10°

C. temperature range.

39. The dynamic flux method of claim 37, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 5°

C. temperature range.

40. The dynamic flux method of claim 37, wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 2.5°

C. temperature range.

43. The method of claim 37, wherein each of the forward and reverse oligonucleotide primers is present in an approximately equimolar concentration.