Digital amplification

• US 8,859,206 B2
• Filed: 03/24/2011
• Issued: 10/14/2014
• Est. Priority Date: 08/02/1999
• Status: Expired due to Fees

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Vogelstein, Bert, Kinzler, Kenneth W.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/071,105
• Publication Number:
  US 20110201004A1
• Time in Patent Office: 1,300 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 quantity of a genetic sequence in a mixed population of human genomic nucleic acid sequences comprising at least a first and a second human genomic sequence, wherein the first sequence is a sequence of a wild-type allele of a locus and a second sequence is a sequence of a mutant allele of the locus, comprising:

distributing or diluting a mixed population of cell-free, human genomic nucleic acid template molecules from a sample in which the fraction of mutant alleles is less than 20%, into a set comprising at least fifteen assay samples such that said at least fifteen assay samples each comprises less than ten template molecules;

amplifying the template molecules in the assay samples, wherein an assay sample with a single template molecule forms homogeneous amplification products in the assay sample;

analyzing by determining nucleic acid sequence of amplification products in the assay samples of the set with homogeneous amplification products to determine a first number of assay samples in the set which contain the first sequence and a second number of assay samples in the set which contain the second sequence;

comparing the first number to the second number to ascertain a ratio which reflects the composition of the mixed population;

identifying a mutation in the mixed population if a statistically significant fraction of assay samples comprises the second sequence.

2. The method of claim 1 wherein the assay samples of the set have on average 0.5 molecules of template.

3. The method of claim 1 wherein between 0.1 and 0.9 of the assay samples yield an amplification product.

4. The method of claim 1 wherein the mixed population of nucleic acid sequences is distributed or diluted to a single template molecule level in the assay samples.

5. The method of claim 1 wherein the mixed population of nucleic acid sequences is from a body sample.

6. The method of claim 5 wherein the mixed population of nucleic acids sequences is from a body sample selected from the group consisting of stool, blood, and lymph nodes.

7. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least twenty assay samples each comprise less than ten template molecules.

8. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least twenty-five assay samples each comprise less than ten template molecules.

9. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least thirty assay samples each comprise less than ten template molecules.

10. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least forty assay samples each comprise less than ten template molecules.

11. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least fifty assay samples each comprise less than ten template molecules.

12. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least seventy-five assay samples each comprise less than ten template molecules.

13. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least one hundred assay samples each comprise less than ten template molecules.

14. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least five hundred assay samples each comprise less than ten template molecules.

15. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least one thousand assay samples each comprise less than ten template molecules.

16. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least one thousand assay samples are distributed or diluted to a single template molecule level.

17. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that at least one thousand assay samples has on average 0.5 molecules of template.

18. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that between 0.1 and 0.9 of at least one thousand assay samples yield an amplification product.

19. The method of claim 1 wherein the mixed population of nucleic acids sequences is distributed or diluted such that one half of at least one thousand assay samples have one template molecule.

20. The method of claim 1 wherein the mutation is a somatic mutation.

21. The method of claim 1 wherein the mutation is a cancer gene mutation.

22. The method of claim 1 wherein the template molecules are from a population of cells which are not purely tumor cells.

23. The method of claim 1 wherein between 1% and 10% of the alleles in said human genomic nucleic acid template molecules are the mutant sequence of the allele.

24. The method of claim 1 wherein the mixed population of nucleic acid sequences is from a tissue.

25. A method for detecting quantity of a genetic sequence in a mixed population of human genomic nucleic acid sequences comprising at least a first and a second human genomic sequence, wherein the first sequence is a sequence of a wild-type allele of a locus and a second sequence is a sequence of a mutant allele of the locus, comprising:

distributing or diluting a mixed population of cell-free, human genomic nucleic acid template molecules into a set comprising at least fifteen assay samples such that said at least fifteen assay samples comprise an average of 0.5 molecules of template per assay sample;

amplifying the template molecules in the assay samples, wherein an assay sample with a single template molecule forms homogeneous amplification products in the assay sample;

analyzing by determining nucleic acid sequence of amplification products in the assay samples of the set with homogeneous amplification products to determine a first number of assay samples in the set which contain the first sequence and a second number of assay samples in the set which contain the second sequence;

comparing the first number to the second number to ascertain a ratio which reflects the composition of the mixed population;

identifying a mutation in the mixed population when a statistically significant fraction of assay samples comprises the second sequence.

26. The method of claim 25 wherein the step of analyzing determines sequence of nucleic acid consisting of the wild-type allele, the mutant allele, or both alleles.

27. The method of claim 25 wherein the alleles are at a single chromosomal locus and nucleic acids consisting of all or part of the locus are analyzed in the step of analyzing.

28. The method of claim 25 wherein the mixed population of nucleic acids sequences is from a body sample selected from the group consisting of stool, blood, and lymph nodes.