Asymmetrical Adapters And Methods Of Use Thereof
First Claim
1. A pair of asymmetrical oligonucleotide tail adapters comprising:
- a) a first oligonucleotide tail adapter comprising a 3′
overhang; and
b) a second oligonucleotide tail adapter comprising a 5′
overhang with at least one blocking group at the 3′
end of the strand that does not comprise the 5′
overhang.
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Abstract
A pair of asymmetrical, partially double-stranded oligonucleotide adapters are provided wherein the pair of adapters comprise a first asymmetrical oligonucleotide adapter comprising a single-stranded 3′ overhang and a second asymmetrical double-stranded oligonucleotide adapter comprising a single-stranded 5′ overhang and at least one blocking group on the strand of said second asymmetrical oligonucleotide adapter that does not comprise the 5′ overhang. Also provided are a pair of double-stranded Y oligonucleotide adapters and a pair of double-stranded bubble oligonucleotide adapters and methods of using said asymmetrical adapters for amplification of at least one double stranded nucleic acid molecule, wherein the amplification produces a plurality of amplified nucleic acid molecules having a different nucleic acid sequence at each end are also described. Also provided is a method for exponentially amplifying one strand in a double-stranded nucleic acid molecule. Also provided are methods for preparing libraries of paired tags using COS-linkers. Also provided are cleavable adapters comprising an affinity tag and a cleavable linkage, wherein cleaving the cleavable linkage produces two complementary ends. Methods of using the cleavable adapters to produce a paired tag library are also described.
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Citations
64 Claims
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1. A pair of asymmetrical oligonucleotide tail adapters comprising:
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a) a first oligonucleotide tail adapter comprising a 3′
overhang; andb) a second oligonucleotide tail adapter comprising a 5′
overhang with at least one blocking group at the 3′
end of the strand that does not comprise the 5′
overhang. - View Dependent Claims (2)
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3. A pair of asymmetrical oligonucleotide tail adapters, comprising:
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a) a first partially double-stranded oligonucleotide tail adapter comprising a ligatable end, and a 3′
single-stranded overhang of at least about 8 nucleotides at the opposite end; andb) a second double-stranded oligonucleotide tail adapter comprising a ligatable end, and a 5′
single-stranded overhang comprising at least about 8 nucleotides at the opposite end, wherein the 3′
end of the strand that does not comprise the 5′
overhang comprises at least one blocking group. - View Dependent Claims (4)
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5. A pair of Y oligonucleotide adapters, comprising:
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a) a first partially double-stranded Y oligonucleotide adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and b) a second partially double-stranded Y oligonucleotide adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides, wherein the nucleic acid sequence of the first and second double-stranded Y oligonucleotide adapters are not identical. - View Dependent Claims (6)
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7. A pair of asymmetrical bubble oligonucleotide adapters, comprising:
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a) a first partially double-stranded bubble oligonucleotide adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; and b) a second partially double-stranded bubble oligonucleotide adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region, wherein the nucleic acid sequence of the first and second asymmetrical bubble oligonucleotide adapters are not identical. - View Dependent Claims (8)
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9. A pair of asymmetrical oligonucleotide adapters comprising:
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a) a first oligonucleotide adapter selected from the group consisting of; (i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′
overhang of at least about 8 nucleotides;(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; and b) a second oligonucleotide adapter selected from the group consisting of; (i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′
overhang of at least about 8 nucleotides, wherein the 3′
end of the strand that does not comprise the 5′
overhang comprises at least one blocking group;(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; wherein the nucleic acid sequence of the first and second double-stranded oligonucleotide adapters are not identical.
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10. A method for exponential amplification of one template strand of at least one double-stranded nucleic acid molecule to produce a plurality of amplified molecules having a different sequence at each end, comprising:
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a) ligating to one end of the double-stranded nucleic acid molecule a first asymmetrical adapter selected from the group consisting of; (i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′
overhang of at least about 8 nucleotides;(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; b) ligating to the other end of the double-stranded nucleic acid molecule a second asymmetrical adapter selected from the group consisting of; (i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′
overhang of at least about 8 nucleotides, wherein the 3′
end of the strand that does not comprise the 5′
overhang comprises at least one blocking group;(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; wherein the nucleic acid sequence of the first and second asymmetrical adapters are not identical, thereby producing an end-linked double-stranded nucleic acid molecule having a first asymmetrical adapter at one end and a second asymmetrical adapter at the other end of the double-stranded nucleic acid molecule; c) amplifying the template strand in an amplification reaction comprising a first primer and a second primer, wherein the template strand is one strand of the end-linked nucleic acid molecule, the amplification reaction comprises; (i) contacting the template strand with a first primer, which is complementary to a first primer binding site in the first asymmetrical adapter in the template strand, under conditions in which the first primer synthesizes a first nucleic acid strand in the amplification reaction, wherein the first nucleic acid strand is complementary to the template strand, and wherein the 3′
end of the first nucleic acid strand comprises a second primer binding site that is complementary to a sequence in the second asymmetrical adapter in the template strand; and(ii) contacting the first nucleic acid strand with a second primer which is complementary to the second primer binding site in the first nucleic acid strand under conditions in which the second primer synthesizes a complementary strand of the first nucleic acid strand, thereby producing a plurality of exponentially amplified molecules having a different sequence at each end.
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11. A method for producing and amplifying a paired tag from a first nucleic acid sequence fragment, without cloning, comprising:
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a) joining the 5′ and
3′
ends of a first nucleic acid sequence fragment via a first linker such that the first linker is located between the 5′
end and the 3′
end of the first nucleic acid sequence fragment thereby producing a circular nucleic acid molecule;b) cleaving the circular nucleic acid molecule, thereby producing a second nucleic acid sequence fragment, wherein a 5′
end tag of the first nucleic acid sequence fragment is joined to a 3′
end tag of the first nucleic acid sequence fragment via the first linker;c) ligating a pair of asymmetrical adapters to the ends of the second nucleic acid sequence fragment, wherein the pair of asymmetrical adapters comprise; (i) a first asymmetrical oligonucleotide adapter selected from the group consisting of; (A) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′
overhang of at least about 8 nucleotides;(B) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (C) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; and (ii) a second asymmetrical oligonucleotide adapter selected from the group consisting of; (A) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′
overhang of at least about 8 nucleotides, wherein the 3′
end of the strand that does not comprise the 5′
overhang comprises at least one blocking group;(B) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (C) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; wherein the nucleic acid sequence of the first and second double-stranded oligonucleotide adapters are not identical, thereby producing an end-linked double-stranded nucleic acid molecule having a first asymmetrical adapter at one end and a second asymmetrical adapter at the other end of the double-stranded nucleic acid molecule; and d) amplifying the template strand in an amplification reaction comprising a first primer and a second primer, wherein the template strand is one strand of the end-linked nucleic acid molecule, the amplification reaction comprises; (i) contacting the template strand with a first primer, which is complementary to a first primer binding site in the first asymmetrical adapter in the template strand, under conditions in which the first primer synthesizes a first nucleic acid strand in the amplification reaction, wherein the first nucleic acid strand is complementary to the template strand, and wherein the 3′
end of the first nucleic acid strand comprises a second primer binding site that is complementary to a sequence in the second asymmetrical adapter in the template strand; and(ii) contacting the first nucleic acid strand with a second primer which is complementary to the second primer binding site in the first nucleic acid strand under conditions in which the second primer synthesizes a complementary strand of the first nucleic acid strand, thereby producing and amplifying a paired tag from a first nucleic acid sequence fragment without cloning.
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12. A method for characterizing a nucleic acid sequence, without cloning, comprising:
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a) fragmenting a nucleic acid sequence thereby producing a plurality of first nucleic acid sequence fragments each having a 5′
end and a 3′
end;b) joining the 5′ and
3′
ends of each first nucleic acid sequence fragment to a first linker such that the first linker is located between the 5′
end and the 3′
end of each first nucleic acid sequence fragment in a circular nucleic acid molecule;c) cleaving the circular nucleic acid molecules, thereby producing a plurality of second nucleic acid sequence fragments wherein a subset of the fragments comprise a paired tag derived from each first nucleic acid sequence fragment joined via the first linker; d) ligating a pair of asymmetrical second adapters to the ends of the second nucleic acid sequence fragment, wherein the pair of asymmetrical adapters comprise; (i) a first asymmetrical oligonucleotide adapter selected from the group consisting of; (A) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′
overhang of at least about 8 nucleotides;(B) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (C) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; and (ii) a second asymmetrical oligonucleotide adapter selected from the group consisting of; (A) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′
overhang of at least about 8 nucleotides, wherein the 3′
end of the strand that does not comprise the 5′
overhang comprises at least one blocking group;(B) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and (C) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; wherein the nucleic acid sequence of the first and second asymmetrical oligonucleotide adapters are not identical, thereby producing an end-linked double-stranded nucleic acid molecule having a first asymmetrical adapter at one end and a second asymmetrical adapter at the other end of the double-stranded nucleic acid molecule; and e) amplifying the template strand in an amplification reaction comprising a first primer and a second primer, wherein the template strand is one strand of the end-linked nucleic acid molecule, the amplification reaction comprises; (i) contacting the template strand with a first primer, which is complementary to a first primer binding site in the first asymmetrical adapter in the template strand, under conditions in which the first primer synthesizes a first nucleic acid strand in the amplification reaction, wherein the first nucleic acid strand is complementary to the template strand, and wherein the 3′
end of the first nucleic acid strand comprises a second primer binding site that is complementary to a sequence in the second asymmetrical adapter in the template strand; and(ii) contacting the first nucleic acid strand with a second primer which is complementary to the second primer binding site in the first nucleic acid strand under conditions in which the second primer synthesizes a complementary strand of the first nucleic acid strand, thereby producing a plurality of amplified second nucleic acid fragments; and f) characterizing the 5′ and
3′
end tags of the plurality of amplified second nucleic acid fragments.
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13. A method for producing a paired end library from a nucleic acid sequence comprising:
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a) fragmenting a nucleic acid sequence to produce a plurality of nucleic acid sequence fragments of an appropriate size for packaging into a lambda bacteriophage head; b) ligating COS-linkers comprising a functional lambda bacteriophage packaging (COS) site to the plurality of nucleic acid sequence fragments under conditions in which a concatemer of nucleic acid sequence fragments and intervening COS linkers is produced; c) packaging individual COS-linked nucleic acid sequence fragments from the concatemer into bacteriophage particles, thereby producing a plurality of packaged, circularized COS-linked nucleic acid sequences, wherein the ends of each nucleic acid sequence fragment are linked by a nicked COS site; d) liberating the circularized COS-linked nucleic acid sequences from the bacteriophage particles under conditions that the nicked COS site remain hybridized; e) sealing the nicked COS site in each circularized COS-linked nucleic acid sequence to produce a plurality of closed circular COS-linked nucleic acid sequences; f) fragmenting said plurality of closed circular COS-linked nucleic acid sequences, thereby producing a paired end library from a nucleic acid sequence comprising COS-linked nucleic acid sequence fragments. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A method for producing a paired end library from a nucleic acid sequence comprising:
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a) fragmenting a nucleic acid sequence to produce a plurality of nucleic acid sequence fragments of an appropriate size for packaging into a lambdoid bacteriophage head; b) ligating COS-linkers to the plurality of nucleic acid sequence fragments under conditions in which a concatemer of nucleic acid sequence fragments and COS linkers is produced, wherein said COS-linkers comprise a functional COS site and two loxP sites flanking the functional COS site; c) packaging individual COS-linked nucleic acid sequence fragments from the concatemer into bacteriophage particles, thereby producing a plurality of packaged, circularized COS-linked nucleic acid sequences, wherein the ends of each nucleic acid sequence fragment are linked by a nicked COS site; d) liberating the circularized COS-linked nucleic acid sequences from the bacteriophage particles under conditions that the nicked COS site remain hybridized; e) sealing the nicked COS site in each circularized COS-linked nucleic acid sequence to produce a plurality of closed circular COS-linked nucleic acid sequences; f) maintaining the plurality of closed circular COS-linked nucleic acid sequences under conditions suitable for intramolecular recombination between the two loxP sites in each closed circular COS-linked nucleic acid sequence, thereby removing the functional COS site from the plurality of closed circular COS-linked nucleic acid sequence fragments, thereby producing a plurality of closed circular lox-linked nucleic acid sequences; and g) fragmenting said plurality of closed circular lox-linked nucleic acid sequences, thereby producing a paired end library from a nucleic acid sequence comprising lox-linked nucleic acid sequence fragments. - View Dependent Claims (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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- 43. A cleavable adapter comprising an affinity tag and a cleavable linkage, wherein cleaving the cleavable linkage produces two complementary ends.
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48. A method for producing a paired tag library from a nucleic acid sequence comprising:
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a) fragmenting a nucleic acid sequence thereby producing a plurality of large nucleic acid sequence fragments of a specific size range; b) introducing onto each end of each nucleic acid sequence fragment a cleavable adapter, wherein the cleavable adapter comprises an affinity tag and a cleavable linkage; c) cleaving the cleavable adapter, thereby producing a plurality of nucleic acid sequence fragments having compatible ends; d) maintaining the nucleic acid sequence fragments having compatible ends under conditions in which the compatible ends intramolecularly ligate, thereby producing a plurality of circularized nucleic acid sequences; e) fragmenting the plurality of circularized nucleic acid sequences, thereby producing a plurality of paired tags comprising a linked 5′
end tag and a 3′
end tag of each nucleic acid sequence fragment,thereby producing a paired tag library from a plurality of large nucleic acid sequence fragments. - View Dependent Claims (49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)
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Specification