Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels

• US 20150119290A1
• Filed: 07/08/2014
• Published: 04/30/2015
• Est. Priority Date: 12/15/2009
• Status: Abandoned Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fodor, Stephen P.A., Fu, Glenn K.
• Application Number:
  US14/326,454
• Publication Number:
  US 20150119290A1
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.

Claims

1. A method for processing a genomic DNA sample, comprising:

(a) hybridizing a genomic sample comprising a population of target DNA molecules with a plurality of primers that comprise;

(i) a 3′

target specific region that hybridizes to a sequence of a target DNA of the plurality of target DNA molecules;

(ii) different label regions, each of which is 5′

to the target-specific region, wherein the label regions comprise nucleotides selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases; and

(iii) a universal primer sequence that is 5′

to each of the label regions,

thereby producing duplexes comprising a target DNA molecule of the population of target DNA molecules and a primer of the plurality of primers;

(b) subjecting the duplexes of step (a) to primer extension to extend the primers in the duplexes, thereby producing a mixture comprising extension products comprising tagged copies of the target DNA molecules,

wherein each of the extension products comprises a label region of the different label regions and the universal primer sequence of the plurality of primers;

(c) removing primers of the plurality of primers that have not been extended in step (b) from the mixture of step (b) or degrading any of the plurality of primers that have not been extended in step (b) from the mixture of step (b); and

(d) amplifying the extension products after step c), thereby producing a plurality of different amplicons, wherein each of the different amplicons comprises multiple copies of the same polynucleotide, and wherein;

(i) the amplifying step is done by polymerase chain reaction using a primer that hybridizes to the universal primer sequence of plurality of primers of step (a); and

(ii) each of at least some of the plurality of different amplicons has a different label region of the label regions.

2. The method of claim 1, wherein step (b) comprises subjecting the duplexes of step (a) to two rounds of prior extension prior to step (c).

3. The method of claim 1, wherein the removing or degrading step (c) is done by immobilizing the plurality of primers on a solid support.

4. The method of claim 3, wherein the plurality of primers have a detectable label.

5. The method of claim 3, wherein the plurality of primers have a biotin label.

6. The method of claim 1, wherein the removing or degrading step (c) comprises degrading the plurality of primers with an exonuclease.

7. The method of claim 1, wherein the label regions comprise at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases is selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases.

8. The method of claim 7, wherein the label regions comprise from 2 to 20 nucleotide bases, wherein each of the 2 to 20 nucleotide bases is selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases.

9. The method of claim 7, wherein the label regions comprise at least 8 nucleotide bases, wherein each of the at least 8 nucleotide bases is selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases.

10. The method of claim 1, wherein the plurality of target DNA molecules are enriched from the genomic sample prior to the hybridizing step (a).

11. The method of claim 1, wherein the genomic sample comprises polynucleotides from tumor cells.

12. The method of claim 1, wherein the genomic sample comprises polynucleotides from bacteria.

13. The method of claim 1, wherein the genomic sample comprises polynucleotides encoding viral epitopes.

14. The method of claim 1, wherein the genomic sample comprises human genomic DNA.

15. The method of claim 1, wherein the label regions can correct estimation errors.

16. The method of claim 1, wherein the genomic sample comprising the population of target DNA molecules is an amplification product.

17. The method of claim 1, wherein the plurality of target DNA molecules are cDNA molecules made by reverse transcription of polynucleotides encoding viral epitopes.

18. A method for processing a genomic DNA sample, comprising:

(a) hybridizing a genomic sample comprising a population of initial target DNA molecules with a population of first primers that comprise;

(i) a 3′

target-specific sequence that hybridizes to a sequence in said initial target DNA molecules,

(ii) different degenerate base region (DBR) sequences, each of which is 5′

to said target-specific sequence, wherein said DBR sequences comprise at least one nucleotide base selected from;

R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof; and

(iii) a generic primer sequence that is 5′

to each of said DBR sequences, thereby producing duplexes comprising an initial target DNA molecule of said population of initial target DNA molecules and a primer of said first primers;

(b) subjecting the duplexes of step (a) to one, two or three rounds of primer extension to extend the population of first primers in said duplexes, thereby producing a mixture comprising tagged copies of said initial target DNA molecules, wherein each of said tagged copies comprises a DBR sequence of said DBR sequences and the generic primer sequence of said first population of primers;

(c) removing any of said population of first primers that have not been extended in step (b) from the mixture of step (b) or inactivating any of said population of first primers that have not been extended in step (b) in the mixture of step (b); and

(d) amplifying said tagged copies of said initial target DNA molecules after step c), thereby producing a plurality of different amplicons, wherein each of the different amplicons comprises multiple copies of the same polynucleotide, and wherein;

i) the amplifying step is done by polymerase chain reaction (PCR) using a primer pair that comprises a generic primer that hybridizes to the complement of the generic primer sequence of the first primers of step (a); and

ii) each of at least some of the different amplicons has a different DBR sequence of said DBR sequences.

19. The method of claim 18, wherein step (b) comprises subjecting the duplexes of step (a) to one round of primer extension prior to step (c).

20. The method of claim 18, wherein step (b) comprises subjecting the duplexes of step (a) to two rounds of primer extension prior to step (c).

21. The method of claim 18, wherein said population of first primers have an affinity tag, and the removing or inactivating step (c) is done by immobilizing said population of first primers on a solid support.

22. The method of claim 18, wherein the removing or inactivating step (c) comprises inactivating said population of first primers using an exonuclease.

23. The method of claim 18, wherein said removing or inactivating step (c) comprises enzymatically modifying said population of first primers.

24. The method of claim 18, wherein said DBR sequences comprises at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases are selected from:

R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.

25. The method of claim 24, wherein the DBR sequences comprise 3 to 10 nucleotide bases, wherein each of the 3 to 10 nucleotide bases is selected from:

R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.

26. The method of claim 24, wherein the DBR sequences comprise 10 or more-nucleotide bases, wherein each of the 10 or more nucleotide bases is selected from:

R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.

27. The method of claim 18, wherein the initial target DNA molecules are enriched from an initial genomic sample prior to the hybridizing step (a).

28. The method of claim 18, wherein the genomic sample comprises polynucleotides from a tumor.

29. The method of claim 18, wherein the genomic sample comprises polynucleotides from a microorganism.

30. The method of claim 18, wherein the genomic sample comprises polynucleotides from a virus.

31. The method of claim 18, wherein the genomic sample comprises human genomic DNA.

32. The method of claim 18, wherein the DBR sequences contain an error correcting code.

33. The method of claim 18, wherein said genomic sample comprising a population of initial target DNA molecules is an amplification product.

34. The method of claim 18, wherein said population of initial target DNA molecules are cDNA molecules made by reverse transcription of a viral genome.