POLYNUCLEOTIDE MAPPING AND SEQUENCING

• US 20110306504A1
• Filed: 11/18/2009
• Published: 12/15/2011
• Est. Priority Date: 11/18/2008
• Status: Active Grant

• Current Assignee: BioNano Genomics, Inc.
• Original Assignee: BioNanomatrix Inc
• Inventors: Cao, Han, Deshpande, Parikshit A., Boyce-Jacino, Michael, Xiao, Ming
• Granted Patent: US 9,181,578 B2
• Time in Patent Office: Days
• US Class Current: 506/6
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• 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 2521/301 Endonuclease
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/133 conformational analysis

Abstract

The present invention provides methods of obtaining structural information about a biopolymer sample. The methods include labeling portions of a biopolymer, such as DNA or RNA, linearizing the biopolymer in some cases, and determining the distance between the labels. The user can then compare different samples'"'"' between-label distances to qualitatively compare different samples and to assay a given sample for additions or deletions of nucleotides in the regions flanked by the labels. The methods also permit sequencing of biopolymers.

Claims

1. A method of assaying for the relative position of one or more exons, comprising:

labeling first and second locations on a biopolymer sample with, respectively, a first and a second label such that the first and second labels flank a first region of the biopolymer sample that includes at least one constant exon; and

correlating the distance between the first and second labels to the presence or relative position of an alternative exon in said first region of the biopolymer.

2. The method of claim 1, wherein the biopolymer comprises DNA complementary to mRNA.

3. The method of claim 1, wherein the first and second labels comprise the same fluorophore.

4. The method of claim 1, wherein the first and second labels comprise different fluorophores.

5. The method of claim 1, wherein correlating the distance between the first and second labels to the presence, absence, or relative position of one or more alternative exons comprises comparing the distance between the first and second labels present on the biopolymer sample to the distance between labels that flank the first region of the biopolymer known not to contain an alternative axon.

6. The method of claim 1, wherein the correlating comprises comparing the distance between the first and second labels to the distance between the first and second locations on a biopolymer lacking an alternative exon between the first and second locations.

7. The method of claim 1, further comprising labeling at least a third location on the biopolymer sample with a third label.

11. The method of claim 1, further comprising linearizing at least a portion of the biopolymer sample.

12. The method of claim 11, wherein the linearized portion of the biopolymer sample comprises at least two labels.

13. The method of claim 1, wherein two labels are separated from one another by from about 30 bp.

14. The method of claim 1, further comprising correlating the relative order of two or more probes to one or more structural characteristics of the biopolymer sample.

8. (canceled)

9. The method of claim 8, further comprising correlating the distance between the third label and the first label, the third label and the second label, or both, to the presence, absence, or relative position of an alternative exon disposed between the third label and the first label, between the third label and the second label, or both.

10. The method of claim 9, where the correlating comprises comparing the distance between the third and first labels, between the third and second labels, or both, to the distance between the third and first labels, between the third and second labels, or both, on a biopolymer lacking an alternative exon between the third and first locations, between the third and second locations, or both.

15. A method of obtaining structural information about a DNA sample, comprising:

nicking a first double-stranded DNA sample with a sequence-specific nicking endonucleoase;

incorporating one or more dye-labeled nucleotides at two or more nicking sites effected by the nicking endonuclease;

linearizing a portion of the first double-stranded DNA sample that includes at least two dye-labeled nucleotides; and

registering the relative positions of two or more labeled dye-labeled nucleotides.

16. The method of claim 15, wherein one or more of the labeled nucleotides comprises a terminator nucleotide.

19. The method of claim 15, further comprising determining the relative position of the first double-stranded DNA sample within a principal double-stranded DNA sample from which the first double-stranded DNA sample was derived.

20. The method of claim 19, wherein the first double-stranded DNA sample was obtained from the principal double-stranded DNA sample by digestion.

21. The method of claim 15, further comprising nicking a second double-stranded DNA sample with a sequence-specific nicking endonucleoase, incorporating one or more dye-labeled nucleotides at two or more nicking sites effected by the nicking endonuclease, linearizing a portion of the second double-stranded DNA sample that includes at least two dye-labeled nucleotides, and registering the relative positions of two or more labeled dye-labeled nucleotides.

23. The method of claim 15, further comprising comparing the relative positions of the two or more dye-labeled nucleotides to the positions of the same dye-labeled nucleotides on a second double-stranded DNA sample contacted with the same nicking endonucleoase.

17. (canceled)

18. (canceled)

22. (canceled)

24. (canceled)

25. A method of obtaining sequence information about a nucleic acid biopolymer, comprising:

binding a first fluorescently labeled sequence specific probe having a first binding sequence to a single-stranded nucleic acid biopolymer;

contacting the single-stranded nucleic acid biopolymer with a first terminator nucleotide bearing a fluorescent label A, with a second terminator nucleotide bearing a fluorescent label B, with a third terminator nucleotide bearing a fluorescent label C, and with a fourth terminator nucleotide bearing a fluorescent label D; and

illuminating the nucleic acid biopolymer so as to determine the presence of, the relative positions of, or both, of the first terminator nucleotide, the second terminator nucleotide, the third terminator nucleotide, the fourth terminator nucleotide, or any combination thereof, adjacent to the first labeled sequence-specific probe.

27. The method of claim 25, wherein at least two of fluorescent label A, fluorescent label B, fluorescent label C, or fluorescent label D have different excitation wavelengths.

28. The method of claim 27, wherein at least one of fluorescent label A, fluorescent label B, fluorescent label C, or fluorescent label D has an excitation wavelength different from the excitation wavelength of the first fluorescently labeled sequence-specific probe.

29. The method of claim 25, further comprising contacting at least four fluorescently labeled probes having, respectively, second, third, fourth, and fifth binding sequences to the single-stranded nucleic acid biopolymer.

30. The method of claim 29, wherein the second binding sequence is constructed by eliminating the base at the 5′

end of the first binding sequence and adding a first replacement base to the 3′

end of the first binding sequence.

31. The method of claim 29, wherein the third binding sequence is constructed by eliminating the base at the 5′

end of the first binding sequence and adding a second replacement base to the 3′

end of the first binding sequence.

32. The method of claim 29, wherein the fourth binding sequence is constructed by eliminating the base at the 5′

end of the first binding sequence and adding a third replacement base to the 3′

end of the first binding sequence.

33. The method of claim 29, wherein the fifth binding sequence is constructed by eliminating the base at the 5′

end of the first binding sequence and adding a fourth replacement base to the 3′

end of the first binding sequence.

34. The method of claim 29, wherein the second fluorescently labeled sequence specific probe has an excitation wavelength different from that of the first fluorescently-labeled sequence-specific probe.

35. The method of claim 29, wherein the second binding sequence differs from the first binding sequence.

37. The method of claim 29, further comprising illuminating the nucleic acid biopolymer so as to determine the presence or relative positions of the first terminator nucleotide, the second terminator nucleotide, the third terminator nucleotide, the fourth terminator nucleotide, or any combination thereof, adjacent to the second labeled sequence-specific probe.

26. (canceled)

36. (canceled)

38. A method of obtaining structural information about a nucleic acid biopolymer, comprising:

(a) contacting a double-stranded biopolymer with a nicking endonuclease so as to give rise to at least two nicking sites;

(b) contacting the at least two nicking sites with a first nucleotide bearing a fluorescent label A;

(c) removing excess first nucleotide;

(d) illuminating the double-stranded biopolymer so as to determine the presence or relative positions of the first nucleotide;

(e) contacting the at least two nicking sites with a second nucleotide bearing a fluorescent label B;

(f) removing excess second nucleotide;

(g) illuminating the double-stranded biopolymer so as to determine the presence or relative positions of the second nucleotide;

(h) contacting the at least two nicking sites with a third nucleotide bearing a fluorescent label C;

(i) removing excess third nucleotide;

(j) illuminating the double-stranded biopolymer so as to determine the presence or relative positions of the third nucleotide;

(k) contacting the at least two nicking sites with a fourth nucleotide bearing a fluorescent label D,

(l) removing excess fourth nucleotide; and

(m) illuminating the double-stranded biopolymer so as to determine the presence or relative positions of the first nucleotide.

39. The method of claim 38, further comprising repeating steps (b) through (m).

40. The method of claim 38, further comprising linearizing a portion of the double-stranded biopolymer including at least two labeled nucleotides and determining the distances between the two or more labeled nucleotides residing within the linearized portion of the double-stranded biopolymer.

42. The method of claim 38, further comprising inducing a second nicking site adjacent to the nucleotide residing at the first nicking site.

43. The method of claim 38, wherein labeled nucleotides fully populate at least one biopolymer region between neighboring nicking sites.

44. The method of claim 38, wherein at least a portion of at least one biopolymer region between neighboring nicking sites is not fully populated with labeled nucleotides.

45. The method of claim 38, further comprising determining the sequence of at least a portion of the biopolymer sample by correlating the order of fluorophores visible under illumination to the nucleotides to which one or more of the fluorophores correspond.

41. (canceled)

46. A kit for performing multiplex hybridization, comprising:

a plurality of hybridization probes each of a different color; and

instructions for applying at least two of the plurality of hybridization probes to a nucleic acid sample and linearizing and imaging at least one of the hybridized nucleic acids