Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels

• US 20110160078A1
• Filed: 12/15/2010
• Published: 06/30/2011
• Est. Priority Date: 12/15/2009
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fu, Glenn K., Fodor, Stephen P.A.
• Granted Patent: US 8,835,358 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 of estimating a number of each of a plurality of different target molecules in a sample comprising:

(a) combining the sample with a collection of labels, wherein the collection of labels comprises multiple copies of each of a plurality of different label sequences;

(b) attaching labels to molecules in a stochastic manner to obtain label-target molecules;

(c) optionally amplifying the label-target molecules;

(d) hybridizing the label-target molecules to an array of probes comprising a plurality of features wherein each feature contains a different probe sequence and the probe sequence in each feature is specific for a different label-target combination, wherein the array comprises a plurality of label/target features for each of a plurality of targets; and

(e) counting the number of different label-target features that are labeled for each different target, wherein the count for each different target is the estimate of the number of occurrences of that target in the sample.

2. The method of claim 1 wherein the collection of labels comprises a number of different label sequences that is at least twice the number of each target molecule to be estimated.

3. The method of claim 1 wherein prior to the hybridizing step (d) the label-target molecules are amplified by PCR so that there are a plurality of copies of each label-target molecule that are hybridized to the array of probes.

4. The method of claim 1 wherein a genomic DNA sample comprises the target molecules, said method further comprising:

fragmenting the genomic DNA sample to obtain fragments comprising target molecules,

ligating a first adaptor comprising a common priming sequence is ligated to a first end of the fragments and a second adaptor comprising a label and a second common priming sequence is ligated to the second end of the fragments, wherein the label sequence is variable and the second common priming sequence is constant, and wherein the label is situated between the target fragment and the second common priming sequence, thereby obtaining adaptor-ligated, labeled fragments;

amplifying said adaptor-ligated, labeled fragments byPCR using a primer or a pair of primers to the first and second common priming sequences; and

thereby obtaining amplified labeled targets.

5. The method of claim 1 wherein the number of different labels is at least an order of magnitude larger than the expected number of target molecules.

6. The method of claim 1 wherein labels are attached to targets by ligation.

7. The method of claim 1 wherein the ratio of target molecule occurrences to number of different labels is 1:

10.

8. The method of claim 1 wherein the ratio of target molecule occurrences to number of different labels is 1:

100.

9. The method of claim 1 wherein the ratio of target molecule occurrences to number of different labels is 1:

1000.

10. The method of claim 1 wherein the ratio of target molecule occurrences to number of different labels is 1:

10,000.

11. An array comprising a plurality of feature sets, including a feature set for each of a plurality of different targets, wherein each feature set comprises a plurality of distinguishable features each feature comprising a plurality of oligonucleotide probes having the same sequence over the length of the probe, and wherein the features of a feature set have a common target specific sequence and a variable label sequence, wherein each different feature in the set has the same target specific sequence and a different label specific sequence and wherein each feature set has a feature for each different label in a set of labels.

12. The array of claim 11 wherein the array has at least three different features sets, each feature set being specific for a single target and having a feature for each possible combination of that target and a label from a label set.

13. The array of claim 11 wherein the array has between 3 and 10 different features sets, each feature set being specific for a single target and having a feature for each possible combination of that target and a label from a label set.

14. The array of claim 11 wherein the array has between 10 and 100 different features sets, each feature set being specific for a single target and having a feature for each possible combination of that target and a label from a label set.

15. The array of claim 11 wherein the array has between 100 and 1000 different features sets, each feature set being specific for a single target and having a feature for each possible combination of that target and a label from a label set.

16. The array of claim 11 wherein the array has between 1000 and 10,000 different features sets, each feature set being specific for a single target and having a feature for each possible combination of that target and a label from a label set.

17. A method of counting a number of target polynucleotides in a sample, the method comprising the steps of:

labeling each target polynucleotide in the sample so that substantially every target polynucleotide is associated with a unique oligonucleotide tag-label, each oligonucleotide tag-label comprising an identification tag;

separating the tagged target polynucleotides from the non-target polynucleotides in the sample and determining the number of different oligonucleotide tags that are attached to each type of target polynucleotide in the sample by determining the number of different identification tag-labels that are attached to target polynucleotides; and

determining the number of target polynucleotides from the number of different oligonucleotide tags in the sample.

18. A method of determining a number of target polynucleotides, the method comprising the steps of:

providing for each target polynucleotide a plurality of nucleic acid probes specific for the target polynucleotide, each nucleic acid probe having a different oligonucleotide tag;

combining in a reaction mixture the plurality of nucleic acid probes with the target polynucleotides so that substantially every target polynucleotide associates with a nucleic acid probe to form a probe-target complex, the plurality of nucleic acid probes having a number sufficiently greater than the number of target polynucleotides so that substantially every probe-target complex has a unique oligonucleotide tag;

treating the sample to enrich the probe-target complexes; and

determining the number of different oligonucleotide tags in a sample of enriched probe-target complexes by hybridization thereby determining the number of target polynucleotide in the mixture.

19. The method of claim 18 wherein the target polynucleotide is a restriction fragment having a first single strand sequence overhang and a second, different single strand overhang, and wherein a first adaptor has a single stranded overhang that is perfectly complementary to the first overhang and a second different adaptor has a single stranded overhang that is perfectly complementary to the second overhang.

20. The method of claim 19 wherein the restriction fragment is formed by digesting genomic DNA with at least one restriction endonuclease.

21. A method of estimating a number of target polynucleotides in a mixture, the method comprising the steps of:

labeling by sampling each target polynucleotide in the mixture so that substantially every target polynucleotide has an oligonucleotide label ligated thereto;

amplifying the oligonucleotide labels that are ligated to target polynucleotides; and

determining the number of different oligonucleotide labels in the amplified sample by hybridization, thereby estimating the number of target polynucleotides in the mixture.

22. The method of claim 21 wherein the step of amplifying comprises PCR.

23. The method of claim 21 wherein the step of amplifying comprises reverse transcription.

24. The method of claim 21 wherein the step of amplifying comprises rolling circle amplification.

25. The method of claim 21 wherein the step of amplifying comprises an isothermal amplification step using a strand displacing DNA polymerase.

26. A method for counting n wherein n is the number of molecules of a first target molecule that are present in a sample comprising:

attaching to each occurrence of the first target molecule a label from a set of diverse labels wherein said set comprises m different labels, thereby generating for each occurrence of the first target molecule, a new molecule that comprises a copy of the first target molecule and a label, wherein more than 90% of the new molecules have a label that is different from the labels on the other new molecules;

detecting each new molecule by detecting each label present on a new molecule;

and counting the number of new molecules, thereby counting the number of molecules of the first target molecule in the sample.

27. The method of claim 26 wherein the ratio of m to n is greater than 50.

28. The method of claim 26 wherein the ratio of m to n is greater than 100.

29. The method of claim 26 wherein the ratio of m to n is greater than 500.

30. The method of claim 26 wherein the ratio of m to n is greater than 1000.