Digital Amplification

• US 20110201004A1
• Filed: 03/24/2011
• Published: 08/18/2011
• Est. Priority Date: 08/02/1999
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: KINZLER, Kenneth W., VOGELSTEIN, Bert
• Granted Patent: US 8,859,206 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The identification of pre-defined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. The exponential, analog nature of the polymerase chain reaction is transformed into a linear, digital signal suitable for this purpose. Single molecules can be isolated by dilution and individually amplified; each product is then separately analyzed for the presence of pre-defined mutations. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.

Claims

1. A method for detecting a cancer-associated mutant nucleic acid that is present in a patient sample at a low level relative to a corresponding wild-type nucleic acid, the method comprising:

diluting nucleic acids in a biological sample to form a set comprising a plurality of assay samples;

amplifying the nucleic acids in the assay samples to form a population of amplified molecules;

performing an assay on the amplified molecules in each assay sample to determine whether a cancer-associated mutation is present in at least one of the assay samples;

wherein the step of diluting in performed until at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than a ratio of the mutant nucleic acid to the wild-type nucleic acid required to detect the mutant nucleic acid if it is present in the assay sample.

2. The method of claim 1 wherein the step of diluting is performed until between 0.1 and 0.9 of the assay samples yield an amplification product when subjected to a polymerase chain reaction.

3. The method of claim 1 wherein the step of diluting is performed until all of the assay samples yield an amplification product when subjected to a polymerase chain reaction and each assay sample contains less than 10 nucleic acid template molecules containing a reference genetic sequence.

4. The method of claim 1 wherein the step of diluting is performed until all of the assay samples yield an amplification product when subjected to a polymerase chain reaction and each assay sample contains less than 100 nucleic acid template molecules containing a reference genetic sequence.

5. The method of claim 1 wherein the biological sample is cell-free.

6. The method of claim 1 wherein the number of assay samples within the set is greater than 10.

7. The method of claim 1 wherein the number of assay samples within the set is greater than 50.

8. The method of claim 1 wherein the number of assay samples within the set is greater than 100.

9. The method of claim 1 wherein the number of assay samples within the set is greater than 500.

10. The method of claim 1 wherein the number of assay samples within the set is greater than 1000.

11. The method of claim 1 wherein the step of amplifying and the step of analyzing are performed on assay samples in the same receptacle.

12. The method of claim 1 wherein a molecular beacon probe is used in the step of analyzing, wherein a molecular beacon probe is an oligonucleotide with a stem-loop structure having a photoluminescent dye at one of the 5′

or 3′

ends and a quenching agent at the opposite 5′

or 3′

end.

13. The method of claim 1 wherein the step of analyzing employs gel electrophoresis.

14. The method of claim 1 wherein the step of analyzing employs hybridization to at least one nucleic acid probe.

15. The method of claim 1 wherein the step of analyzing employs hybridization to at least two nucleic acid probe.

16. The method of claim 12 wherein two molecular beacon probes are used, each having a different photoluminescent dye.

17. The method of claim 12 wherein the molecular beacon probe detects a wild-type nucleic acid better than a mutant nucleic acid.

18. The method of claim 1 wherein the step of amplifying employs a single pair of primers.

19. The method of claim 1 wherein the step of amplifying employs a polymerase which is activated only after heating.

20. The method of claim 1 wherein the step of amplifying employs at least 40 cycles of heating and cooling.

21. The method of claim 1 wherein the step of amplifying employs at least 50 cycles of heating and cooling.

22. The method of claim 1 wherein the step of amplifying employs at least 60 cycles of heating and cooling.

23. The method of claim 1 wherein the biological sample is selected from the group consisting of stool, blood, and lymph nodes.

24. The method of claim 1 wherein the biological sample is blood or bone marrow of a leukemia or lymphoma patient who has received anti-cancer therapy.

25. The method of claim 1 wherein the mutant nucleic acid is a translocated allele

26. The method of claim 1 wherein the mutant nucleic acid is within an amplicon which is amplified during neoplastic development.

27. The method of claim 1 wherein the mutant nucleic acid is a rare exon sequence

28. The method of claim 1 wherein the nucleic acids being analyzed comprise cDNA of RNA transcripts.

29. A method for determining the ratio of a selected genetic sequence in a population of genetic sequences from a blood sample, comprising the steps of:

diluting nucleic acid template molecules from a blood sample to form a set comprising a plurality of assay samples;

amplifying the template molecules within the assay samples to form a population of amplified molecules in the assay samples of the set;

analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence;

comparing the first number to the second number to ascertain a ratio which reflects the composition of the blood sample.

30. The method of claim 29 wherein the step of amplifying employs real-time polymerase chain reactions.

31. The method of claim 30 wherein the real-time polymerase chain reactions comprise a dual-labeled fluorogenic probe.

32. The method of claim 29 further comprising the step of:

identifying an allelic imbalance based on the ratio ascertained.

33. The method of claim 29 wherein the selected genetic sequences and the reference genetic sequence are non-polymorphic markers.

34. The method of claim 29 wherein the selected genetic sequence and the reference genetic sequence are on distinct chromosomes.

35. A method for determining the ratio of a selected non-polymorphic marker in a population of genetic sequences in a biological sample, comprising the steps of:

diluting nucleic acid template molecules in a biological sample to form a set comprising a plurality of assay samples;

amplifying the template molecules within the assay samples to form a population of amplified molecules in the assay samples of the set;

analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected non-polymorphic marker and a second number of assay samples which contain a reference non-polymorphic marker, wherein the selected and reference non-polymorphic markers are on distinct chromosomes;

comparing the first number to the second number to ascertain a ratio which reflects the composition of the biological sample; and

identifying an allelic imbalance based on the ratio ascertained.

36. The method of claim 35 wherein the biological sample is a blood sample.

37. The method of claim 35 wherein the step of amplifying employs real-time polymerase chain reactions.

38. The method of claim 37 wherein the real-time polymerase chain reactions comprise a dual-labeled fluorogenic probe.

39. A method for determining the ratio of a selected genetic sequence in a population of genetic sequences from a blood sample, comprising the steps of:

amplifying template molecules within a set comprising a plurality of assay samples to form a population of amplified molecules in each of the assay samples of the set, wherein the template molecules are obtained from a blood sample;

analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence, wherein at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than the ratio of selected genetic sequences to total genetic sequences required to determine the presence of the selected genetic sequence;

comparing the first number to the second number to ascertain a ratio which reflects the composition of the blood sample.

40. The method of claim 39 wherein the step of amplifying employs real-time polymerase chain reactions.

41. The method of claim 40 wherein the real-time polymerase chain reactions comprise a dual-labeled fluorogenic probe.

42. The method of claim 39 further comprising the step of:

identifying an allelic imbalance based on the ratio ascertained.

43. The method of claim 39 wherein the selected genetic sequences and the reference genetic sequence are non-polymorphic markers.

44. The method of claim 39 wherein the selected genetic sequence and the reference genetic sequence are on distinct chromosomes.

45. A method for determining the ratio of a selected non-polymorphic marker in a population of non-polymorphic markers from a biological sample, comprising the steps of:

amplifying template molecules within a set comprising a plurality of assay samples to form a population of amplified molecules in each of the assay samples of the set, wherein the template molecules are obtained from a biological sample;

analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected non-polymorphic marker and a second number of assay samples which contain a reference non-polymorphic marker, wherein at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than the ratio of selected non-polymorphic marker to total non-polymorphic markers required to determine the presence of the selected non-polymorphic marker, wherein the selected genetic sequence and the reference genetic sequence are on distinct chromosomes;

comparing the first number to the second number to ascertain a ratio which reflects the composition of the biological sample; and

identifying an allelic imbalance based on the ratio ascertained.

46. The method of claim 45 wherein the step of amplifying employs real-time polymerase chain reactions.

47. The method of claim 46 wherein the real-time polymerase chain reactions comprise a dual-labeled fluorogenic probe.

48. The method of claim 45 wherein the biological sample is from blood.