Methods of determining polymorphisms

• US 10,100,349 B2
• Filed: 09/30/2014
• Issued: 10/16/2018
• Est. Priority Date: 09/30/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Macleod, Iain J., Rowley, Christopher F., Essex, Max
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/026,031
• Publication Number:
  US 20160244817A1
• Time in Patent Office: 1,477 Days
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Methods and compositions for determining the presence of a polymorphism at a target nucleotide position in a plurality of target nucleic acid sequences is provided.

Claims

1. A method of determining the presence of a polymorphism at one or more of a target nucleotide position in a plurality of target nucleic acid sequences, with each target nucleic acid sequence having a sense strand sequence and an antisense strand sequence, wherein the one or more of a target nucleotide position is flanked by a 5′

flanking region and a 3′

flanking region in each of the sense strand sequence and the antisense strand sequence comprising

amplifying the target nucleic acid sequences to produce amplicons using a forward primer and a reverse primer, wherein the forward primer includes a 3′

adaptor region, wherein the 3′

adaptor region is a determined consensus sequence for the 5′

flanking region of the sense strand,

wherein the reverse primer includes a 3′

adaptor region, wherein the 3′

adaptor region is complementary to a determined consensus sequence for the 3′

flanking region of the sense strand,

wherein the amplicons have a sense strand amplicon and an antisense strand amplicon,

wherein the sense strand amplicon includes a probe binding site including one or more of a target nucleotide at the one or more of a target nucleotide position with a 5′

flanking region being the 3′

adapter region sequence of the forward primer and with a 3′

flanking region being the complement of the 3′

adapter region sequence of the reverse primer,

wherein the antisense strand amplicon includes a probe binding site including one or more of a target nucleotide at the one or more of a target nucleotide position with a 5′

flanking region being the 3′

adapter region sequence of the reverse primer and with a 3′

flanking region being the complement of the 3′

adaptor region of the forward primer,

contacting the amplicons with sense-oriented nucleic acid probes or antisense-oriented nucleic acid probes including a label and having a probe sequence identical to the complement of the probe binding site, and

detecting the label of hybridized nucleic acid probes.

2. The method of claim 1, wherein the forward primer further includes a pan degenerate region.

3. The method of claim 1 wherein the reverse primer further includes a pan degenerate region.

4. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids, bacterial nucleic acids, or fungal nucleic acids.

5. The method of claim 4 wherein the viral nucleic acids are viral RNA or viral DNA.

6. The method of claim 4 wherein the bacterial nucleic acids are bacterial RNA or bacterial DNA.

7. The method of claim 4 wherein the fungal nucleic acids are fungal RNA or fungal DNA.

8. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids including wild type viral nucleic acids with a wild type nucleotide at the one or more of a target nucleotide position and mutated viral nucleic acids with the polymorphism at the one or more of a target nucleotide position.

9. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids including wild type viral nucleic acids with a wild type nucleotide at the one or more of a target nucleotide position and mutated viral nucleic acids with the polymorphism at the one or more of a target nucleotide position, wherein the polymorphism provides drug resistance.

10. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids including wild type viral nucleic acids with a wild type nucleotide at the one or more of a target nucleotide position and mutated viral nucleic acids with the polymorphism at the one or more of a target nucleotide position, and wherein the sense oriented nucleic acid probes or the antisense oriented nucleic acid probes include wild type probes with a first label and mutant detecting probes with a second label and where the first label and the second label are spectrally resolvable.

11. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids including wild type viral nucleic acids with a wild type nucleotide at the one or more of a target nucleotide position and mutated viral nucleic acids with the polymorphism at the one or more of a target nucleotide position, and wherein the sense oriented nucleic acid probes or the antisense oriented nucleic acid probes include wild type probes with a first label and mutant detecting probes with a second label and where the first label and the second label are spectrally resolvable, wherein the method further includes determining the relative amount of the wild type viral nucleic acids versus the mutated viral nucleic acids by comparing the detected labels.

12. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids including wild type viral nucleic acids with a wild type nucleotide at the one or more of a target nucleotide position and mutated viral nucleic acids with the polymorphism at the one or more of a target nucleotide position, and wherein the sense oriented nucleic acid probes or the antisense oriented nucleic acid probes include wild type probes with a first label and mutant detecting probes with a second label and where the first label and the second label are spectrally resolvable, wherein the method further includes determining the relative amount of the wild type viral nucleic acids versus the mutated viral nucleic acids by comparing the detected labels, wherein the relative amount is indicative of drug resistance of the population of viral nucleic acids.

13. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids including wild type viral nucleic acids with a wild type nucleotide at the one or more of a target nucleotide position and mutated viral nucleic acid with the polymorphism at the one or more of a target nucleotide position, and wherein the sense oriented nucleic acid probes or the antisense oriented nucleic acid probes include wild type probes with a first label and mutant detecting probes with a second label and where the first label and the second label are spectrally resolvable, wherein the method further includes determining the relative amount of the wild type viral nucleic acids versus the mutated viral nucleic acids by comparing the detected labels, wherein the relative amount is indicative of the presence of virulent versus non-virulent strains of virus.

14. The method of claim 1 wherein the plurality of target nucleotide sequences is a population of viral nucleic acids with first viral nucleic acids having a first nucleotide at the one or more of a target nucleotide position indicative of a non-virulent strain of virus and mutated viral nucleic acids with the polymorphism at the one or more of a target nucleotide position indicative of a virulent strain of virus, and wherein the sense oriented nucleic acid probes or the antisense oriented nucleic acid probes include mutant detecting probes with a first label and wherein detecting the first label is indicative of the presence of the virulent strain of virus.

15. The method of claim 1 wherein the forward primers and the reverse primers have constant adaptor regions and variable pan degenerate regions.

16. The method of claim 1 wherein the nucleic acid probes include molecular beacons, hydrolysis probes, locked nucleic acid probes, or FRET probes.

17. The method of claim 1 wherein the plurality of target nucleotide sequences comprises a population of HIV viral DNA, Newcastle disease viral DNA, or hepatitis C viral DNA.

18. The method of claim 1 wherein the polymorphism at the one or more of a target nucleotide position is a single nucleotide polymorphism.

19. The method of claim 1 wherein the 5′

flanking region to the one or more of a target nucleotide position is between about 1 to about 10 nucleotides in length and wherein the 3′

flanking region to the one or more of a target nucleotide position is between about 1 to about 10 nucleotides in length.

20. The method of claim 1 wherein the 5′

flanking region to the one or more of a target nucleotide position is between about 3 to about 8 nucleotides in length and wherein the 3′

flanking region to the one or more of a target nucleotide position is between about 3 to about 8 nucleotides in length.

21. The method of claim 1 wherein the 5′

flanking region to the one or more of a target nucleotide position is between about 6 to about 8 nucleotides in length and wherein the 3′

flanking region to the one or more of a target nucleotide position is between about 6 to about 8 nucleotides in length.

22. The method of claim 1 wherein the 5′

flanking region to the one or more of a target nucleotide position is about 7 nucleotides in length and wherein the 3′

flanking region to the one or more of a target nucleotide position is about 7 nucleotides in length.

23. The method of claim 1 wherein the sense oriented nucleic acid probe or the antisense oriented nucleic acid probe includes a complement to the nucleotide at the one or more of a target nucleotide position which is flanked by a 5′

flanking region and a 3′

flanking region.

24. The method of claim 1 wherein the sense oriented nucleic acid probe or the antisense oriented nucleic acid probe is an exact complement of the probe binding site of the amplicons.

25. The method of claim 1 wherein the sense oriented nucleic acid probe or the antisense oriented nucleic acid probe is about 10 to about 20 nucleotides in length.

26. The method of claim 1 wherein the sense oriented nucleic acid probe or the antisense oriented nucleic acid probe is about 12 to about 15 nucleotides in length.

27. The method of claim 1 wherein the sense oriented nucleic acid probe or the antisense oriented nucleic acid probe is about 15 nucleotides in length.

28. The method of claim 2 wherein the pan degenerate region is between about 25 to about 35 nucleotides in length.

29. The method of claim 2 wherein the pan degenerate region is between about 28 to about 32 nucleotides in length.

30. The method of claim 2 wherein the pan degenerate region is about 30 nucleotides in length.

31. The method of claim 1 wherein the label is a fluorescent label.

45. The method of claim 3 wherein the pan degenerate region is between about 25 to about 35 nucleotides in length.

46. The method of claim 3 wherein the pan degenerate region is between about 28 to about 32 nucleotides in length.

47. The method of claim 3 wherein the pan degenerate region is about 30 nucleotides in length.

32. A method of mutating flanking regions of one or more of a target nucleotide position in a plurality of target nucleic acid sequences to provide a known probe binding site on amplicons, with each target nucleic acid sequence having a sense strand sequence and an antisense strand sequence, wherein the one or more of a target nucleotide position is flanked by a 5′

flanking region and a 3′

flanking region in each of the sense strand sequence and the antisense strand sequence comprising

amplifying the target nucleic acid sequence to produce the amplicons using a forward primer and a reverse primer, wherein the forward primer includes a 3′

adaptor region, wherein the 3′

adaptor region is a determined consensus sequence for the 5′

flanking region of the sense strand,

wherein the reverse primer includes a 3′

adaptor region, wherein the 3′

adaptor region is complementary to a determined consensus sequence for the 3′

flanking region of the sense strand,

wherein the amplicons have the known probe binding site determined by the determined consensus sequences from the forward and reverse primers and the target nucleotide at the one or more of a target nucleotide position.

33. The method of claim 32 wherein a polymorphism is present at the one or more of a target nucleotide position.

34. The method of claim 32 wherein a single nucleotide polymorphism is present at the one or more of a target nucleotide position.

35. A method of providing a known probe binding site sequence including one or more of a target nucleotide in amplicons of a plurality of target nucleic acid sequences, with each target nucleic acid sequence having a sense strand sequence and an antisense strand sequence, wherein one or more of a target nucleotide position is flanked by a 5′

flanking region and a 3′

flanking region in each of the sense strand sequence and the antisense strand sequence comprising

amplifying the target nucleic acid sequence to produce the amplicons using a forward primer and a reverse primer, wherein the forward primer includes a 3′

adaptor region, wherein the 3′

adaptor region is a determined consensus sequence for the 5′

flanking region of the sense strand,

wherein the reverse primer includes a 3′

adaptor region, wherein the 3′

adaptor region is complementary to a determined consensus sequence for the 3′

flanking region of the sense strand,

wherein the known probe binding site of the amplicons is determined by the determined consensus sequences from the forward and reverse primers and the target nucleotide at the one or more of a target nucleotide position.

36. The method of claim 35, wherein the forward primer further includes a pan degenerate region.

37. The method of claim 35 wherein the reverse primer further includes a pan degenerate region.

38. The method of claim 35 wherein the one or more of a target nucleotide is a polymorphism.

39. The method of claim 35 wherein the one or more of a target nucleotide is a single nucleotide polymorphism.

40. A method of removing secondary polymorphisms flanking one or more of a target nucleotide position in a plurality of target nucleic acid sequences to provide a known probe binding site in amplicons, with each target nucleic acid sequence having a sense strand sequence and an antisense strand sequence, wherein the target nucleotide position is flanked by a 5′

flanking region and a 3′

flanking region in each of the sense strand sequence and the antisense strand sequence comprising

amplifying the target nucleic acid sequence to produce the amplicons using a forward primer and a reverse primer, wherein the forward primer includes a 3′

adaptor region, wherein the 3′

adaptor region is a determined consensus sequence for the 5′

flanking region of the sense strand,

wherein the reverse primer includes a 3′

adaptor region, wherein the 3′

adaptor region is complementary to a determined consensus sequence for the 3′

flanking region of the sense strand,

wherein the amplicons have the known probe binding site determined by the determined consensus sequences from the forward and reverse primers and the target nucleotide at the one or more of a target nucleotide position.

41. The method of claim 40, wherein the forward primer further includes a pan degenerate region.

42. The method of claim 40 wherein the reverse primer further includes a pan degenerate region.

43. The method of claim 40 wherein a polymorphism is present at the one or more of a target nucleotide position.

44. The method of claim 40 wherein a single nucleotide polymorphism is present at the one or more of a target nucleotide position.