Molecular counting

• US 20070172873A1
• Filed: 01/22/2007
• Published: 07/26/2007
• Est. Priority Date: 01/23/2006
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Macevicz, Stephen, Brenner, Sydney
• Granted Patent: US 7,537,897 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The invention provides methods and compositions for counting molecules in a sample, wherein each molecule is labeled with a unique oligonucleotide tag. Such tags are amplified and identified rather than the molecules themselves; that is, the problem of counting molecules is converted into the problem of counting tags. In one aspect of the invention, molecules to be counted are labeled by sampling. That is, conjugates are formed between the molecules to be counted and oligonucleotide tags of a very large set, or repertoire. After conjugation, a sample of conjugates is taken that is sufficiently small so that substantially every molecule has a unique oligonucleotide tag. Counting of different tags may be accomplished in a variety of ways. In one aspect, different tags may be counted by carrying out a series of sorting steps to generate successively less complex mixtures in which tags are enumerated using length-encoded “metric” tags. In another aspect, different tags may be counted by directly sequencing a sample of tags using any one of several different sequencing methodologies.

Claims

1. A method of determining a number of target molecules in a sample, the method comprising the steps of:

(a) providing molecule-tag conjugates each comprising an oligonucleotide tag such that substantially every different molecule of the sample is attached to a different oligonucleotide tag, each oligonucleotide tag comprising a concatenation of subunits selected from a set of subunits, each subunit being a different nucleotide or oligonucleotide and having a position, and the set of subunits having a size of from 2 to 6 members;

(b) dividing the oligonucleotide tags of the molecule-tag conjugates into aliquots by sorting the oligonucleotide tags according to the identity of a subunit within a first or a successive position; and

(c) repeating step (b) for at least one aliquot in each successive application of step (b) until at least one aliquot has no oligonucleotide tags that can be separated into aliquots and determining the number of molecules in the sample from the number of times step (b) has been applied.

2. The method of claim 1 wherein said number of molecules in said sample is within a range determined by a first number equal to the size of the subset taken to a power equal to the lowest number of said times said step (b) has been applied to produce an aliquot having no oligonucleotide tags less one and a second number equal to the size of the subset taken to a power equal to the greatest number of times said step (b) has been applied to produce an aliquot having no oligonucleotide tags less one.

3. The method of claim 1 wherein said step (c) is carried out for a plurality of said aliquots in at least one application of said step (b) so that a plurality of aliquots each have no oligonucleotide tags that can be separated into aliquots and wherein said number of molecules in said sample is determined from the numbers of times said step (b) has been applied in each of such aliquots.

4. The method of claim 3 wherein said molecules are polynucleotides and said oligonucleotide tags are binary tags.

5. The method of claim 4 wherein said binary tags each comprise a concatenate of dinucleotide subunits.

6. The method of claim 1 wherein said step of dividing includes the steps of extending a primer annealed to said oligonucleotide tags to incorporate either a first terminator having a capture moiety whenever the first terminator is complementary to a nucleotide of a subunit of a oligonucleotide tag or a second terminator having a capture moiety whenever the second terminator is complementary to a nucleotide of a subunit of a oligonucleotide tag, capturing oligonucleotide tags having primers with a first terminator by a capture agent that specifically binds to the capture moiety and melting the captured oligonucleotide tags to form a first aliquot, capturing oligonucleotide tags having primers with a second terminator by a capture agent that specifically binds to the capture moiety and melting the captured oligonucleotide tags to form a second aliquot.

7. The method of claim 6 wherein said captured oligonucleotide tags are replicated after said step of melting.

8. A method of counting a number of target polynucleotides in a specimen, the method comprising the steps of:

labeling by sampling each target polynucleotide in the specimen so that substantially every target polynucleotide is associated with a unique oligonucleotide tag, each oligonucleotide tag comprising a sorting tag and a identification tag;

successively sorting the oligonucleotide tags a number of times by their sorting tags to form one or more separate mixtures; and

determining the number of different oligonucleotide tags in at least one of the one or more separate mixtures by the identification tags thereof; and

determining the number of target polynucleotides from the number of successive sortings and the number of different oligonucleotide tags in the one or more separate mixtures.

9. The method of claim 8 wherein said sorting tags are binary tags.

10. The method of claim 9 wherein said binary tags are provided in a number and said identification tags are provided in a number, and wherein the number of binary tags is substantially larger than the number of identification tags.

11. The method of claim 10 wherein said identification tags are metric tags.

12. 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 polynucleotides associates with a nucleic acid probe to form a selected nucleic acid probe that is resistant to at least one nuclease activity, the plurality of nucleic acid probes having a size sufficiently greater than the number of target polynucleotides so that substantially every selected nucleic acid probe has a unique oligonucleotide tag;

isolating the selected nucleic acid probes by treating the reaction mixture with one or more nuclease activities; and

determining nucleotide sequences of oligonucleotide tags in a sample of isolated selected nucleic acid probes to determine the number of different oligonucleotide tags therein, thereby determining the number of target polynucleotide in the mixture.

13. The method of claim 12 wherein said target polynucleotide is a restriction fragment having at least one unique sequence overhang and wherein each of said nucleic acid probes is an adaptor having an overhang on each end, one adaptor overhang being complementary to one overhang of the restriction fragment and another adaptor overhang being complementary to another overhang of the restriction fragment so that upon ligation a double stranded DNA circle is formed.

14. The method of claim 13 wherein said restriction fragment is formed by digesting genomic DNA with at least one type IIs restriction endonuclease.

15. The method of claim 13 wherein said step of determining further includes forming one or more concatenates of oligonucleotide tags from said sample and sequencing the one or more concatenates.

16. The method of claim 13 wherein said at least one type IIs restriction endonuclease is a double cleavage type IIs restriction endonuclease.

17. 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 a unique oligonucleotide tag;

amplifying the oligonucleotide tags of the labeled target polynucleotides; and

determining the number of different oligonucleotide tags in a sample of amplified oligonucleotide tags by determining nucleotide sequences thereof, thereby estimating the number of target polynucleotides in the mixture.

18. The method of claim 17 wherein said step of amplifying is carried out by emulsion PCR.

19. The method of claim 18 wherein said step of determining is carried out by a DNA sequencing technology selected from the group consisting of pyrosequencing, Sanger-based sequencing, and ligation-based sequencing.

20. 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 polynucleotides associates with a nucleic acid probe to form a selected nucleic acid probe capable of isolation from other nucleic acids of the reaction mixture, the plurality of nucleic acid probes having a size sufficiently greater than the number of target polynucleotides so that substantially every selected nucleic acid probe has a unique oligonucleotide tag;

isolating the selected nucleic acid probes; and

determining nucleotide sequences of oligonucleotide tags in a sample of isolated selected nucleic acid probes to determine the number of different oligonucleotide tags therein, thereby determining the number of target polynucleotide in the mixture.

21. The method of claim 20 wherein said nucleic acid probes are padlock probes and wherein said step of isolating includes treating said reaction mixture with one or more nuclease activities.

22. The method of claim 20 wherein said nucleic acid probes are ligation probes each having a first component and a second component that when ligated together form a ligation product capable of amplification and wherein said step of isolating includes amplifying the ligation product to form a capturable amplicon that can be separated from said reaction mixture.

23. A set of oligonucleotide tags for enumerating molecular events, the set comprising:

a plurality of oligonucleotides each having a length within the range of from 6 to 32 nucleotides, wherein the length of each oligonucleotide in the plurality is the same and wherein each oligonucleotide has a sequence with no homopolymeric regions having a length greater than three.

24. The set of oligonucleotide tags of claim 23 wherein said sequence has no homopolymeric regions greater having said length greater than two.

25. The set of oligonucleotide tags of claim 24 wherein said sequence has no homopolymeric regions.

26. The set of oligonucleotide tags of claim 24 wherein said sequence has a GC content of less than or equal to sixty percent.

27. The set of oligonucleotide tags of claim 24 wherein said sequence has no hairpin sequences.

28. The set of oligonucleotide tags of claim 24 wherein said sequence has a parity value.

29. The set of oligonucleotide tags of claim 24 wherein (i) said length of each of said oligonucleotide is at least eight nucleotides, (ii) said plurality is at least 1000 and (iii) said sequences of said oligonucleotides are maximally different.

30. The set of oligonucleotides of claim 24 wherein (i) said length of each of said oligonucleotide is at least eight nucleotides, (ii) said plurality is at least 1000 and (iii) said sequence of each of said oligonucleotides differs from that of every other said oligonucleotide in said plurality by at least three nucleotides.

31. 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 a unique metric tag;

amplifying the metric tags of the labeled target polynucleotides;

separating the amplified metric tags to form a separation profile of distinct bands; and

counting the number of distinct bands of metric tags, thereby estimating the number of target polynucleotides in the mixture.