METHODS AND COMPOSITIONS FOR THE ANALYSIS OF BIOLOGICAL MOLECULES

• US 20110124518A1
• Filed: 01/16/2009
• Published: 05/26/2011
• Est. Priority Date: 01/17/2008
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Cantor, Charles R.
• Granted Patent: US 8,852,864 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals

Abstract

Provided herein are compositions and methods for analysis of nucleic acids, including, methods and compositions for genotyping, haplotyping, sequencing and performing other genetic and epigenetic analyses on nucleic acids, for example. In some embodiments, methods and compositions suitable for whole-genome sequencing on single molecules of nucleic acid are provided. In some embodiments, analysis of single molecules of nucleic acid are performed in conjunction with nanopores and/or nanopore devices.

Claims

1. A method for analyzing a nucleic acid, which comprises:

(a) contacting a target nucleic acid with a plurality of nucleic acid probes under conditions in which probes having a nucleotide sequence substantially complementary to a subsequence of the target nucleic acid can hybridize to the target nucleic acid (“

complementary nucleotide sequence”

), wherein;

(i) the complementary nucleotide sequence is located at the 5′

or 3′

end of each probe;

(ii) the ends of each complementary nucleotide sequence of each probe in probe pairs are (1) adjacent to one another when hybridized to the target nucleic acid, (2) are adjacent to an intervening linker probe, (3) do not abut one another and intervening nucleotides are added by an enzyme capable of polymerizing nucleotides complementary to the target nucleic acid sequence;

(iii) each probe comprises a detector region having a polynucleotide sequence not substantially complementary to a subsequence of the target nucleic acid;

(iv) each detector region is located at the 5′

or 3′

end of each probe and on the end of the probe opposite the complementary nucleotide sequence;

(v) the end of a detector region of a first probe of a probe pair is in proximity to the end of a detector region of a second probe in another probe pair, with the proviso that the end of a detector region of one probe in two probe pairs is not in proximity to the end of the detector region of another probe; and

(vi) the first probe flanks the second probe when the first probe and second probe are hybridized to the target nucleic acid;

(b) ligating the ends of the of the complementary nucleotide sequences of probe pairs that are adjacent to one another when hybridized to the target nucleic acid;

(c) joining the ends of detector regions in proximity to one another, thereby forming a linked probe molecule;

(d) passing the linked probe molecule through the pore of a nanopore device; and

(e) determining the base sequence of the linked probe molecule, whereby the target nucleic acid is analyzed.

2. The method of claim 1, wherein the target nucleic acid is analyzed by a method selected from the group consisting of:

nucleic acid sequencing;

copy number determination;

methylation analysis;

genotyping;

haplotyping;

detection of specific sequences;

identification of insertions, deletions, or translocations;

identification of single nucleotide polymorphisms and sequence variations;

detection of allelic variance;

measurement of phenotypic variance, and combinations thereof.

3. The method of claim 1, wherein the target nucleic acid comprises DNA.

4. The method of claim 3, wherein the target nucleic acid consists of DNA.

5. The method of claim 1, wherein the target nucleic acid is from a single sample.

6. The method of claim 1, wherein the target nucleic acid comprises pooled DNA.

7. The method of claim 6, wherein the target nucleic acid consists of pooled DNA.

8. The method of claim 1, wherein the target nucleic acid is fragmented DNA.

9. The method of claim 1, wherein the target nucleic acid is methylated DNA.

10. The method of claim 9, wherein the target nucleic acid is treated with an agent that converts non-methylated nucleotide, or methylated nucleotide, in the target nucleic acid to a detectable entity.

11. The method according to claim 10, wherein the target nucleic acid is treated with an agent that converts non-methylated cytosine to uracil.

12. The method of claim 1, wherein the target nucleic acid comprises RNA.

13. The method of claim 12, wherein the target nucleic acid consists of RNA.

14. The method of claim 1, wherein the target nucleic acid comprises pooled RNA.

15. The method of claim 14, wherein the target nucleic acid consists of pooled RNA.

16. The method of claim 1, wherein the probe comprises DNA.

17. The method of claim 16, wherein the probe consists of DNA

18. The method of claim 1, wherein the probe comprises RNA.

19. The method of claim 18, wherein the probe consists of RNA.

20. The method of claim 1, wherein the probe comprises PNA.

21. The method of claim 20, wherein the probe consists of PNA.

22. The method of claim 1, wherein the detector region of the probe comprises a detectable moiety.

23. The method of claim 22, wherein the detectable moiety is selected from the group consisting of:

a radioactive agent, a fluorescent agent, a light scattering agent, a molecular beacon, an affinity capture agent, a chemiluminescent agent, a protein agent, a peptide agent, a chromogenic agent, a biomolecule, and a combination thereof.

24. The method of claim 1, wherein the detector portions of probes in proximity to one another are joined by chemical ligation.

25. The method of claim 1, wherein the detector portions of probes in proximity to one another are joined by cross-linking.

26. The method of claim 25, wherein the cross-linking is effected by UV light, a non-specific cross-linking agent, a sequence specific cross-linking agent, or a combination thereof.

27. The method of claim 1, wherein the linked probe molecule is passed through a nanopore device by a gradient.

28. The method of claim 27, wherein the gradient is an electrical gradient, chemical gradient, magnetic gradient, or a combination thereof.

29. The method of claim 1, wherein the base sequence of linked probes is determined by a nucleotide sequencing method.

30. The method of claim 29, wherein the nucleotide sequencing method is pyrosequencing.

31. The method of claim 29, wherein the nucleotide sequencing method is sequencing by synthesis.