METHODS, COMPOSITIONS AND KITS FOR GENERATION OF STRANDED RNA OR DNA LIBRARIES
First Claim
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1. A method for generating a directional cDNA library, the method comprising:
- a) annealing one or more primers to a template RNA;
b) extending the one or more primers in the presence of a reaction mixture comprising dATP, dCTP, dGTP, dTTP, and dUTP, wherein the reaction mixture comprises a ratio of dUTP to dTTP, wherein the ratio permits incorporation of dUTP at a desired density, thereby generating a one or more first strand complementary DNAs (cDNAs) comprising dUTP incorporated at a desired density;
c) selectively cleaving the one or more first strand cDNAs comprising dUTPs incorporated at a desired density with uracil-N-glycosylase (UNG) and an agent capable of cleaving a phosphodiester backbone at an abasic site created by the UNG, wherein the cleaving generates a plurality of first strand cDNA fragments of a desired size comprising a blocked 3′
end;
d) annealing a first adapter comprising a partial duplex and a 3′
overhang to a 3′
end of one or more of the plurality of first strand cDNA fragments comprising a blocked 3′
end, wherein the first adapter comprises a sequence A, and wherein the annealing comprises hybridizing a random sequence at the 3′
overhang to a complementary sequence present at the 3′
end of the one or more of the plurality of first strand cDNA fragments comprising a blocked 3′
end;
e) extending the 3′
overhang hybridized to the complementary sequence with a DNA polymerase, wherein one or more double stranded cDNA fragments comprising the sequence A at one end is generated;
f) ligating a second adapter comprising a sequence B to the one or more double stranded cDNA fragments comprising the sequence A at one end, wherein the ligating generates one or more double stranded cDNA fragments comprising the sequence A at one end and the sequence B at an opposite end, thereby generating the directional polynucleotide library; and
g) optionally, amplifying and/or sequencing the directional cDNA library.
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Abstract
The invention provides methods and compositions, including kits, for the construction of directional nucleic acid libraries. The invention further provides methods and compositions for the amplification and sequencing of directional cDNA libraries.
72 Citations
41 Claims
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1. A method for generating a directional cDNA library, the method comprising:
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a) annealing one or more primers to a template RNA; b) extending the one or more primers in the presence of a reaction mixture comprising dATP, dCTP, dGTP, dTTP, and dUTP, wherein the reaction mixture comprises a ratio of dUTP to dTTP, wherein the ratio permits incorporation of dUTP at a desired density, thereby generating a one or more first strand complementary DNAs (cDNAs) comprising dUTP incorporated at a desired density; c) selectively cleaving the one or more first strand cDNAs comprising dUTPs incorporated at a desired density with uracil-N-glycosylase (UNG) and an agent capable of cleaving a phosphodiester backbone at an abasic site created by the UNG, wherein the cleaving generates a plurality of first strand cDNA fragments of a desired size comprising a blocked 3′
end;d) annealing a first adapter comprising a partial duplex and a 3′
overhang to a 3′
end of one or more of the plurality of first strand cDNA fragments comprising a blocked 3′
end, wherein the first adapter comprises a sequence A, and wherein the annealing comprises hybridizing a random sequence at the 3′
overhang to a complementary sequence present at the 3′
end of the one or more of the plurality of first strand cDNA fragments comprising a blocked 3′
end;e) extending the 3′
overhang hybridized to the complementary sequence with a DNA polymerase, wherein one or more double stranded cDNA fragments comprising the sequence A at one end is generated;f) ligating a second adapter comprising a sequence B to the one or more double stranded cDNA fragments comprising the sequence A at one end, wherein the ligating generates one or more double stranded cDNA fragments comprising the sequence A at one end and the sequence B at an opposite end, thereby generating the directional polynucleotide library; and g) optionally, amplifying and/or sequencing the directional cDNA library. - View Dependent Claims (5, 7, 8, 9, 10, 11, 14, 21, 36)
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2. (canceled)
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3. A method for generating a directional cDNA library, the method comprising:
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a) treating a template dsDNA with a nicking enzyme, wherein the treating generates one or more breaks in a phosphodiester backbone of one strand of the template dsDNA, wherein the break produces one or more 3′
hydroxyls in the one strand;b) extending the one or more 3′
hydroxyls, wherein the extending is performed in the presence of a reaction mixture comprising dATP, dCTP, dGTP, dTTP, and dUTP, wherein the reaction mixture comprises a ratio of dUTP to dTTP, wherein the ratio permits incorporation of dUTP at a desired density, thereby generating one or more first strand complementary DNAs (cDNAs) comprising dUTP incorporated at a desired density;c) selectively cleaving the one or more first strand cDNAs comprising dUTPs incorporated at a desired density with uracil-N-glycosylase (UNG) and an agent capable of cleaving a phosphodiester backbone at an abasic site created by the UNG, wherein the cleaving generates a plurality of first strand cDNA fragments of a desired size comprising a blocked 3′
end;d) annealing a first adapter comprising a partial duplex and a 3′
overhang to a 3′
end of one or more of the plurality of first strand cDNA fragments comprising a blocked 3′
end, wherein the first adapter comprises a sequence A, and wherein the annealing comprises hybridizing a random sequence at the 3′
overhang to a complementary sequence present at the 3′
end of the one or more of the plurality of first strand cDNA fragments comprising a blocked 3′
end;e) extending the 3′
overhang hybridized to the complementary sequence with a DNA polymerase, wherein one or more double stranded cDNA fragments comprising the sequence A at one end is generated;f) ligating a second adapter comprising a sequence B to the one or more double stranded cDNA fragments comprising the sequence A at one end, wherein the ligating generates one or more double stranded cDNA fragments comprising the sequence A at one end and the sequence B at an opposite end thereby generating a directional cDNA library; and g) optionally, amplifying and/or sequencing the directional cDNA library. - View Dependent Claims (16, 18, 22, 23, 24, 25, 26, 27, 38, 39, 40, 41)
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4. A method for generating a whole genome library, the method comprising:
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a) denaturing nicked and/or fragmented dsDNA template nucleic acid; b) annealing a first adapter comprising a partial duplex and a 3′
overhang to a 3′
end of one or more of the plurality of single-stranded DNA fragments, wherein the first adapter comprises a sequence A, and wherein the annealing comprises hybridizing a random sequence at the 3′
overhang to a complementary sequence present at the 3′
end of the one or more of the plurality of single-stranded DNA fragments;c) extending the 3′
overhang hybridized to the complementary sequence with a DNA polymerase, wherein one or more double stranded cDNA fragments comprising the sequence A at one end is generated;d) ligating a second adapter comprising a sequence B to the one or more double stranded cDNA fragments comprising the sequence A at one end, wherein the ligating generates one or more double stranded cDNA fragments comprising the sequence A at one end and the sequence B at an opposite end thereby generating a directional cDNA library; and e) optionally, amplifying and/or sequencing the directional cDNA library.
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6. (canceled)
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12. (canceled)
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13. (canceled)
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15. (canceled)
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17. (canceled)
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19. (canceled)
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20. (canceled)
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28-35. -35. (canceled)
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37. (canceled)
Specification