Methods and compositions for the analysis of biological molecules

• US 10,557,164 B2
• Filed: 09/17/2014
• Issued: 02/11/2020
• 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.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US14/488,757
• Publication Number:
  US 20150004610A1
• Time in Patent Office: 1,973 Days
• Field of Search: None
• 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 target nucleic acid in a sample, which comprises:

(a) hybridizing a plurality of nucleic acid probes to the target nucleic acid, wherein each probe comprises a target binding region having a polynucleotide sequence complementary to the target nucleic acid and a non-target binding detector region having a polynucleotide sequence not complementary to the target nucleic acid wherein such polynucleotide sequence provides information about the chromosomal location of the target, wherein;

i) the plurality of nucleic acid probes comprise probe pairs,

ii) the end of the complementary nucleotide sequence of one probe is adjacent to the end of the other probe in the same probe pair when hybridized to the target nucleic acid,

iii) each detector region is located on the end of the probe opposite the complementary nucleotide sequence,

iv) the end of the detector region of a first probe of a probe pair is in proximity to the end of the detector region of a second probe of a different 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

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

(b) joining the ends of the complementary nucleotide sequences of the probes in the same probe pair when hybridized to the target nucleic acid,

by ligation, if there are no bases there between the ends of the complementary nucleotide sequences of the probes in the same probe pair, or

by extension followed by ligation, if there are one or more bases there between the ends of the complementary nucleotide sequences of the probes in the same probe pair,

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

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

2. The method of claim 1, wherein the detector region of at least one probe further comprises a signal generating moiety.

3. The method of claim 2, wherein the signal generating moiety comprises at least one of a fluorescent agent, a light scattering agent, a molecular beacon, an affinity capture agent, a nucleotide, a copolymer, a sugar, a protein, a peptide, a mass tag, a charge tag, a hydrophobic tag, a volatile tag, and a combination thereof.

4. The method of claim 2, wherein the signal generating moiety comprises a fluorescent agent.

5. The method of claim 1, wherein the plurality of probes are a library of greater than 10 probes.

6. The method of claim 5, wherein the library comprises more than 100 probes.

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

8. The method of claim 1, wherein a labeled nucleotide or nucleotides are used when joining the ends of the complementary nucleotide sequences of the probes in the same probe pair by extension.

9. The method of claim 1, wherein the linked probe molecule is detected by passage through a nanopore.

10. A method for determining the haplotype of a target nucleic acid in a sample, which comprises:

(a) hybridizing a first probe and second probe to a first portion of the target, wherein the first probe is 5′

of the second probe, and hybridizing a third and fourth probe to a second portion of the target, wherein the third probe is 5′

to the fourth probe, wherein at least one of the first or second probes and at least one of the third and fourth probes comprise a region that does not hybridize to the target, and wherein the 3′

end of the first probe or the 5′

end of the second probe is adjacent to a first sequence variation of the target sequence, and the 3′

end of the third probe or the 5′

end of the fourth probe is adjacent to a second variation of the target sequence,

wherein the first probe comprises a detector region at the 5′ and

the second probe comprising a detector region at the 3′

, wherein the third probe comprises a detector region at the 5′ and

the fourth probe comprising a detector region at the 3′

;

(b) ligating the first and second probes and the third and fourth probes;

(c) joining the end of the detector region of the second probe with the end of the detector region of the third probe, thereby forming a linked probe molecule;

(d) separating the linked probes from the nucleic acid comprising the target; and

(e) correlating detection of the linked probes with a haplotype of the target nucleic acid.

11. The method of claim 10, wherein the linked probes are analyzed by passage through a nanopore.

12. The method of claim 10, wherein the portion of the probe that does not hybridize to the target comprise a signal generating moiety.

13. The method of claim 12, wherein the signal generating moiety comprises at least one of a fluorescent agent, a light scattering agent, a molecular beacon, an affinity capture agent, a nucleotide, a copolymer, a sugar, a protein, a peptide, a mass tag, a charge tag, a hydrophobic tag, a volatile tag, and a combination thereof.