SELECTION OF NUCLEIC ACIDS BY SOLUTION HYBRIDIZATION TO OLIGONUCLEOTIDE BAITS

• US 20100029498A1
• Filed: 02/04/2009
• Published: 02/04/2010
• Est. Priority Date: 02/04/2008
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, Whitehead Institute for Biomedical Research
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Nusbaum, Chad, Lander, Eric S., Gnirke, Andreas
• Application Number:
  US12/365,650
• Publication Number:
  US 20100029498A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction

Abstract

Methods of selection of nucleic acids using solution hybridization, methods of sequencing nucleic acids including such selection methods, and products for use in the methods are disclosed.

Claims

1. A method for solution-based selection of nucleic acids comprising hybridizing in solution (1) a group of nucleic acids and (2) a set of bait sequences, to form a hybridization mixture,

contacting the hybridization mixture with a molecule or particle that binds to or is capable of separating the set of bait sequences from the hybridization mixture, and

separating the set of bait sequences from the hybridization mixture to isolate a subgroup of nucleic acids that hybridize to the bait sequences from the group of nucleic acids, wherein the subgroup of nucleic acids is a part or all of a set of target sequences that is desired to be selected,

wherein the sequence composition of the set of bait sequences determines the nucleic acids directly selected from the group of nucleic acids.

2. The method of claim 1, wherein the set of bait sequences comprises an affinity tag on each bait sequence.

3. The method of claim 2, wherein the affinity tag is a biotin molecule or a hapten.

4. The method of claim 1, wherein the molecule or particle that binds to or is capable of separating the set of bait sequences from the hybridization mixture binds to the affinity tag.

5. The method of claim 4, wherein the molecule or particle that binds to or is capable of separating the set of bait sequences is an avidin molecule, or an antibody that binds to the hapten or an antigen-binding fragment thereof.

9. The method of claim 1, wherein the bait sequences are oligonucleotides between about 100 nucleotides and 300 nucleotides in length.

12. The method of claim 1, wherein the bait sequences are oligonucleotides between about 300 nucleotides and 1000 nucleotides in length.

13. The method of claim 1, wherein the target-specific sequences in the oligonucleotides are between about 40 and 1000 nucleotides in length.

27. The method of claim 1, wherein the group of nucleic acids is fragmented genomic DNA.

52. The method of claim 1, wherein the number of bait sequences in the set of bait sequences is greater than 1,000.

63. The method of claim 1, further comprising subjecting the isolated subgroup of nucleic acids to one or more additional rounds of solution hybridization with the set of bait sequences.

64. The method of claim 1, further comprising subjecting the isolated subgroup of nucleic acids to one or more additional rounds of solution hybridization with a different set of bait sequences.

65. The method of claim 1, wherein the group of nucleic acids consists of RNA or cDNA derived from RNA.

71. The method of claim 1, wherein the molarity of at least 50% of the isolated subgroup of nucleic acids is within 20-fold of the mean molarity.

74. The method of claim 1, wherein at least 50% of the bases in the isolated subgroup of nucleic acids are present at and can achieve sequence coverage with at least 50% of the mean averaged over all target bases.

77. A method of sequencing or resequencing nucleic acids comprising,

isolating by solution hybridization a subgroup of nucleic acids according to claim 1, and

subjecting the isolated subgroup of nucleic acids to nucleic acid sequencing.

78. A method of genotyping nucleic acids comprising,

isolating by solution hybridization a subgroup of nucleic acids according to claim 1, and

subjecting the isolated subgroup of nucleic acids to genotyping.

102. A method for determining the presence or sequence of a nucleic acid sequence, cell, tissue or organism in a sample, comprising

obtaining a sample containing nucleic acids,

subjecting the nucleic acids in the sample to solution-based selection of nucleic acids according to claim 1, and

determining the presence or sequence of one or more nucleic acids of the subgroup of nucleic acids obtained by selection, whereby the presence or sequence of the one or more nucleic acids indicates the presence of a nucleic acid sequence, cell, tissue or organism in the sample.

6-8. -8. (canceled)

10. (canceled)

11. (canceled)

14-26. -26. (canceled)

28-51. -51. (canceled)

53-62. -62. (canceled)

66-70. -70. (canceled)

72. (canceled)

73. (canceled)

75. (canceled)

76. (canceled)

79. A method of producing a set of bait sequences comprising

obtaining a pool of synthetic long oligonucleotides, originally synthesized on a microarray and

amplifying the oligonucleotides to produce a set of bait sequences.

97. A method of producing a set of RNA bait sequences comprising

producing a set of bait sequences according to claim 79,

adding a RNA polymerase promoter sequence at the ends of the bait sequences, and

synthesizing RNA sequences using RNA polymerase.

80-96. -96. (canceled)

98-101. -101. (canceled)

103-109. -109. (canceled)