Methods of screening nucleic acids for single nucleotide variations
First Claim
1. A method of detecting a nucleotide variation within a nucleic acid of interest comprising:
- (a) a step comprising generating a set of extension products from a first nucleic acid and causing modified nucleotides to be present in the extension products, wherein the modified nucleotides comprise a single type of base,(b) a step comprising hybridizing the extension products to a second nucleic acid,(c) a step comprising contacting the hybridizing nucleic acid with one or more agents that collectively remove the modified nucleotides,(d) a step comprising contacting the hybridizing nucleic acid with a first enzyme that extends the extension products in the presence of three types of nuclease-resistant nucleotides and not in the presence of nucleotides that comprise the same type of base as the modified nucleotides, wherein each of the three types of nuclease-resistant nucleotides comprises a different type of base,(e) a step comprising contacting the hybridizing nucleic acid with a second enzyme that removes nucleotides from the 3′
end of the extension products, wherein the second enzyme does not remove the nuclease-resistant nucleotides,(f) a step comprising contacting the hybridizing nucleic acid with a third enzyme that extends the extension products in the presence of nucleotides,(g) a step comprising distinguishing those extension products comprising a nuclease-resistant nucleotide from those extension products not comprising a nuclease-resistant nucleotide, wherein the first nucleic acid is a reference nucleic acid and the second nucleic acid is the nucleic acid of interest or wherein the first nucleic acid is the nucleic acid of interest and the second nucleic acid is a reference nucleic acid, thereby detecting nucleotide variation in the nucleic acid of interest.
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Accused Products
Abstract
Disclosed are methods and compositions for detecting variation in nucleic acids. The disclosed method compares the sequence of a nucleic acid of interest with the sequence of a reference nucleic acid to sensitively identify variations between the sequence of a nucleic acid of interest and the sequence of a reference nucleic acid. The disclosed method generally involves excision and replacement of selected nucleotides in nucleic acid strands hybridized to other strands. In the method, if the excised nucleotide was mismatched with the nucleotide in the other, hybridized strand, then the replacement nucleotide will not be mismatched. If the excised nucleotide was not mismatched with the nucleotide in the other, hybridized strand, then the excised nucleotide is not replaced. This difference allows detection of variation in the nucleic acid of interest. In some forms of the method, by replacing excised nucleotides with nuclease-resistant nucleotides, strands in which excised nucleotides are replaced will be resistant to nuclease digestion while strands in which excised nucleotides are not replaced will be sensitive to nuclease digestion. By exposing the hybridizing nucleic acids to nuclease following replacement of excised nucleotides, the strands in which excised nucleotides are not replaced can be destroyed by the nuclease while strands in which excised nucleotides are replaced can be preserved. The remaining strands can then be detected and whether the strand survived nuclease digestion can be noted. Strands that survive nuclease digestion are indicative of the presence of variation in the nucleic acid of interest.
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Citations
46 Claims
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1. A method of detecting a nucleotide variation within a nucleic acid of interest comprising:
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(a) a step comprising generating a set of extension products from a first nucleic acid and causing modified nucleotides to be present in the extension products, wherein the modified nucleotides comprise a single type of base, (b) a step comprising hybridizing the extension products to a second nucleic acid, (c) a step comprising contacting the hybridizing nucleic acid with one or more agents that collectively remove the modified nucleotides, (d) a step comprising contacting the hybridizing nucleic acid with a first enzyme that extends the extension products in the presence of three types of nuclease-resistant nucleotides and not in the presence of nucleotides that comprise the same type of base as the modified nucleotides, wherein each of the three types of nuclease-resistant nucleotides comprises a different type of base, (e) a step comprising contacting the hybridizing nucleic acid with a second enzyme that removes nucleotides from the 3′
end of the extension products, wherein the second enzyme does not remove the nuclease-resistant nucleotides,(f) a step comprising contacting the hybridizing nucleic acid with a third enzyme that extends the extension products in the presence of nucleotides, (g) a step comprising distinguishing those extension products comprising a nuclease-resistant nucleotide from those extension products not comprising a nuclease-resistant nucleotide, wherein the first nucleic acid is a reference nucleic acid and the second nucleic acid is the nucleic acid of interest or wherein the first nucleic acid is the nucleic acid of interest and the second nucleic acid is a reference nucleic acid, thereby detecting nucleotide variation in the nucleic acid of interest. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method of detecting a nucleotide variation within a first nucleic acid comprising:
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a. a step comprising generating a set of extension products from a reference nucleic acid in the presence of modified nucleotides comprising a single type of base, wherein the extension products incorporate the modified nucleotides, b. a step comprising hybridizing the extension products to the first nucleic acid, c. a step comprising contacting the hybridizing nucleic acid with one or more agents that collectively remove the modified nucleotides, d. a step comprising contacting the hybridizing nucleic acid with a first enzyme that extends the extension products in the presence of three types of labeled nucleotides and not in the presence of nucleotides that comprise the same type of base as the modified nucleotides, wherein each of the three types of labeled nucleotides comprises a different type of base, e. a step comprising distinguishing those extension products comprising a labeled nucleotide from those extension products not comprising a labeled nucleotide, thereby detecting nucleotide variation in the first nucleic acid.
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Specification