Methods of nucleic acid sample preparation
First Claim
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1. A method of preparing nucleic acids for analysis, the method comprising:
- (a) adding one or more nucleotides to a 3′
end of a double-stranded nucleic acid comprising a target nucleotide sequence, wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;
(b) ligating an adapter nucleic acid to the double-stranded nucleic acid to which the capture moiety modified nucleotide has been added to produce a ligation product, wherein a sequence of one or more nucleotides at a 3′
end of the adapter nucleic acid is complementary with the one or more nucleotides added to the 3′
end of the double-stranded nucleic acid in step (a);
(c) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety modified nucleotide; and
(d) amplifying the ligation product by polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid.
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Abstract
Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.
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Citations
27 Claims
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1. A method of preparing nucleic acids for analysis, the method comprising:
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(a) adding one or more nucleotides to a 3′
end of a double-stranded nucleic acid comprising a target nucleotide sequence, wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;(b) ligating an adapter nucleic acid to the double-stranded nucleic acid to which the capture moiety modified nucleotide has been added to produce a ligation product, wherein a sequence of one or more nucleotides at a 3′
end of the adapter nucleic acid is complementary with the one or more nucleotides added to the 3′
end of the double-stranded nucleic acid in step (a);(c) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety modified nucleotide; and (d) amplifying the ligation product by polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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18. A method of preparing nucleic acids for analysis, the method comprising:
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(a) preparing a cDNA by conducting a randomly-primed first strand synthesis reaction using a nucleic acid preparation comprising an RNA as a template and a second strand synthesis reaction using a product of the randomly-primed first strand synthesis reaction as a template, wherein the RNA comprises a target nucleotide sequence; (b) end repairing the cDNA to produce a blunt-ended, double-stranded nucleic acid comprising the target nucleotide sequence; (c) washing the blunt-ended, double-stranded nucleic acid; (d) adding one or more nucleotides to the 3′
end of the nucleic acid washed in step (c), wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;(e) washing the nucleic acid produced in step (d); (f) ligating an adapter nucleic acid that comprises a ligatable duplex portion and an overhang sequence to the nucleic acid washed in step (e) to produce a ligation product, wherein the overhang sequence is complementary with the one or more nucleotides added in step (d); (g) amplifying the ligation product by polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid; (h) amplifying an amplification product of step (g) by polymerase chain reaction using a second adapter primer and a second target-specific primer, wherein the second target-specific primer is nested relative to the first target-specific primer; and (i) washing the amplification product of step (h). - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification