Methods of specifically labeling nucleic acids using CRISPR/Cas
First Claim
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1. A method of labeling a nucleic acid sequence comprising:
- a) contacting genomic DNA with a guide RNA having a seed sequence which is complementary to a target sequence in the genomic DNA and with Cas9 nickase to produce a nick in the genomic DNA at a specific location adjacent to the target sequence; and
contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked genomic DNA at the specific location; and
b) contacting the nicked genomic DNA with a motif-specific nicking endonuclease thereby producing a second nick in the nicked genomic DNA at a motif sequence; and
contacting the nicked genomic DNA with a polymerase and second fluorescently labeled nucleotide of the same color or different color, wherein the second fluorescently labeled nucleotide is incorporated into the nicked genomic DNA at the motif sequence location;
wherein nick-labeled DNA is produced; and
wherein the nick-labeled DNA is detected via fluorescent label.
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Abstract
Provided herein are methods of detecting a target nucleic acid sequence. In one embodiment, the method includes contacting genomic DNA with a guide RNA having a portion complementary to the target sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at a specific location adjacent to the target sequence. The method further includes contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide. The fluorescently labeled nucleotide is incorporated into the nicked DNA at the specific location and the target nucleic acid sequence is detected via fluorescent label.
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Citations
20 Claims
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1. A method of labeling a nucleic acid sequence comprising:
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a) contacting genomic DNA with a guide RNA having a seed sequence which is complementary to a target sequence in the genomic DNA and with Cas9 nickase to produce a nick in the genomic DNA at a specific location adjacent to the target sequence; and
contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked genomic DNA at the specific location; andb) contacting the nicked genomic DNA with a motif-specific nicking endonuclease thereby producing a second nick in the nicked genomic DNA at a motif sequence; and
contacting the nicked genomic DNA with a polymerase and second fluorescently labeled nucleotide of the same color or different color, wherein the second fluorescently labeled nucleotide is incorporated into the nicked genomic DNA at the motif sequence location;wherein nick-labeled DNA is produced; and wherein the nick-labeled DNA is detected via fluorescent label. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification