MULTIPLEX NUCLEIC ACID DETECTION METHODS AND SYSTEMS

• US 20120088684A1
• Filed: 10/03/2011
• Published: 04/12/2012
• Est. Priority Date: 04/03/2009
• Status: Active Grant

• Current Assignee: LifeOS Genomics Corporation
• Original Assignee: Timothy Z. Liu
• Inventors: Liu, Timothy Z.
• Granted Patent: US 8,603,743 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/153 Viscosity
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention relates to methods and systems for single molecule based clonal amplification and subsequent detection of nucleic acid molecules, and particularly to the determination of SNPs, mutations, and to the diagnosis of diseases associated with the changes of these nucleic acid molecules.

Claims

1. A method for determining the identity code of identity sequence tags, comprising the steps of:

(a) immobilizing a plurality of analyte molecules on a surface, wherein each analyte molecule comprises an identity sequence (IS) tag that is accessible to a probe for hybridization;

(b) hybridizing a pair of labeled IS probes (LISPs) from a pool of LISPs with said IS tags at base interrogation positions, thereby said pair of LISPs are juxtaposed, wherein each said LISP comprises;

(i) a sequence complimentary to the IS tag sequence it hybridizes to, and (ii) a label associated with a designated base at said base interrogation positions;

(c) performing a paired-probe ligation for ligating said pair of juxtaposed LISPs;

(d) detecting the presence of said labels on said ligated pair of LISPs on said IS tags of said analyte molecules, and elucidating the base composition at said base interrogation positions of said IS tags according to the label combination of said ligated pair of LISPs;

(e) denaturing said ligated pair of LISPs from said analyte molecule;

(f) repeating steps (b) to (e) until all base positions in said IS tags are interrogated without duplication and all base composition of said IS tags are elucidated; and

(g) determining the ID codes of said IS tags on said analyte molecules by comparing said base composition to designated ID codes.

2. The method of claim 1, wherein two specified base positions on said IS tags and one on each side of said paired probes are interrogated.

3. The method of claim 1, wherein there are two sets of probes in each pool of LISPs, with one set being the 3′

labeled IS probes (3′

-LISPs) that comprise 5′

phosphate groups and 3′

labels, and the other set being the 5′

labeled IS probes (5′

-LISPs) that comprises 5′

labels and 3′

hydroxyl group.

4. The method of claim 1, wherein there are four different labels on both sets of LISPs with each label representing a designated base (A, C, G, T) at said interrogation positions, and wherein all probes needed for all possible base combinations at said interrogation positions are included in each pool of LISPs.

5. The method of claim 1, wherein a different pool of LISPs is used in each ligation cycle, and wherein different base positions on said IS tags are sequentially interrogated in each ligation cycle.

6. The method of claim 1, wherein the labels on said LISP comprises fluorescent dyes, electrochemical labels, or nanoparticles.

7. The method of claim 1, wherein said base interrogation positions are within 5 bases from the ligation point of each LISP to maintain a base recognition specificity in said paired-probe ligation.

8. The method of claim 1, wherein said base interrogation positions are within 3 bases from a ligation point of each LISP to maintain a base recognition specificity in said paired-probe ligation.

9. A method of analyzing target nucleic acid sequences in a sample, comprising:

(a) generating from a sample a plurality of first template molecules and a plurality of second template molecules, wherein said first template molecules comprise a sequence of a first target nucleic acid and a first identity sequence (IS) tag, and said second template molecules comprise a sequence of a second target nucleic acid and a second IS tag, and wherein said first IS tag comprises a first identification (ID) code and said second IS tag comprises a second ID code;

(b) generating a first cluster of clonal amplicons by clonal amplification of said first template molecules and a second cluster of clonal amplicons by clonal amplification of said second template molecules, wherein said first cluster of clonal amplicons and said second cluster of clonal amplicons are located spatially separately; and

(c) identifying said ID codes of said IS tags of said clonal amplicons to determine the target nucleic acid sequences represented by said clonal amplicons.

10. The method of claim 9, further comprising determining the corresponding sequence status of said target nucleic acid sequences by analyzing the sequence status of said first cluster of the clonal amplicons and said second cluster of the clonal amplicons.

11. The method of claim 9, further comprising quantifying the number of the clusters of clonal amplicons from each template molecule to infer the amount of each target nucleic acid sequence in said sample.

12. The method of claim 11, further comprising quantifying the reference sequences in said sample.

13. The method of claim 9, further comprising immobilizing said first and second clusters of clonal amplicons on a surface.

14. The method of claim 9, wherein said first and second target nucleic acid sequences can be the same, or can be different, and there can be more than said first and second target nucleic acid sequences.

15. The method of claim 9, wherein said target nucleic acid sequences are selected from the group consisting of genomic DNA, cDNA, and RNA.

16. The method of claim 9, wherein said first and second template molecules are generated through an enzymatic reaction, using primers comprising said IS tags and sequences that are complimentary to at least part of said target nucleic acid sequences, wherein the quantity ratios of the two target nucleic acid sequences in said sample are preserved.

17. The method of claim 13, wherein said first and second clusters of clonal amplicons are generated on said surface through enzymatic amplification or replication, wherein at least two of the primers used in said amplification or replication are attached to said surface and spatially separated during said clonal amplification.

18. The method of claim 9, wherein said clonal amplification is carried out in a plurality of aqueous droplets, each droplet comprising:

reagents for DNA amplification, including a plurality of primers; and

a microparticle with at least one of said primers attached on its surface; and

said template molecule.

19. The method of claim 18, wherein said aqueous droplets are formed by water-in-oil emulsion and are contained in a reaction vessel, wherein said reaction vessel contains an oil phase comprising a water immiscible liquid.

20. The method of claim 18, wherein said DNA amplification is polymerase chain reaction (PCR), and said reaction vessel is thermal cycled during said clonal amplification.

21. The method of claim 18, wherein said clonal amplification of template molecules is carried out on hydrophilic reaction spots patterned on an otherwise hydrophobic surface, wherein each hydrophilic reaction site comprises a said aqueous droplet.

22. The method of claim 21, wherein said hydrophilic reaction sites are covered by a water immiscible liquid to prevent evaporation of aqueous phase and to isolate each individual hydrophilic reaction sites during said clonal amplification.

23. The method of claim 21, wherein said clonal amplification is by polymerase chain reaction (PCR), and said surface is thermal cycled during said clonal amplification.

24. The method of claim 9, wherein said clonal amplification is carried out through circularization of said single stranded template molecules and subsequent isothermal rolling circle amplification (RCA) by a DNA polymerase, on extension oligonucleotides attached at 5′

ends on a surface, wherein said extension oligonucleotides are complementary at 3′

ends to the circularized template molecules and comprise free 3′

—

OH groups for enzymatic extension.

25. The method of claim 24, wherein at least two copies of said circularized template molecule sequences are generated through the enzymatic extension.

26. The method of claim 24, wherein said DNA polymerase comprises φ

29 DNA polymerase.

27. The method of claim 24, wherein the circularization of the template molecule on the extension oligonucleotide is carried out by ligating the two ends of the template molecules that contain unique nucleic acid sequences that are a part of the primer sequences used in producing the template molecule from said target nucleic acid sequence.

28. The method of claim 27, wherein said unique nucleic acid sequences at the two ends of each template molecule are the same, or different, for at least two of the different template molecules.

29. The method of claim 24, wherein said circularized template molecules are between 40 to 400 bases in length.

30. The method of claim 24, wherein said circularized template molecules are between 60 to 200 bases in length.

31. The method of claim 18, wherein said microparticle is a magnetic microparticle.

32. The method of claim 13, wherein said clonal amplicons are attached to the surface of a magnetic microparticle, and said magnetic microparticle is immobilized on said surface by a physical force or by a chemical linkage.

33. The method of claim 32, wherein said physical force is magnetic field.

34. The method of claim 9, wherein the ID codes of said clonal amplicons are simultaneously determined by sequential paired-probe ligation cycles of labeled probes, or by DNA sequencing technologies, and said ID codes reveal the representation of target nucleic acid sequences by each cluster of clonal amplicons.

35. A kit for multiplex nucleic acid analysis, comprising:

primers for enzymatic reactions, wherein some of the primers comprise a plurality of identity sequence (IS) tags, and pools of labeled probes, wherein said labeled probes comprise labeled IS probes (LISPs).

36. The kit of claim 35, wherein each pool of labeled probes comprises two sets of LISPs, one set being 3′

-LISPs, and the other set being 5′

-LISPs, and each set of LISPs comprises a specified base interrogation position on all probes.

37. The kit of claim 35, wherein there are four different labels on each set of LISPs with each label representing a designated base (A, C, G, T) at said interrogation position, and all probes needed for all possible base combinations at said interrogation position are included in each pool of LISPs.