Methods of specifically labeling nucleic acids using crispr/cas

A method of detecting the length of an individual telomere, the method comprising:

• a) contacting genomic DNA with a guide RNA having a portion complementary to the telomere repeat sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the telomere repeat sequence;
• b) contacting the genomic DNA with a second nicking endonuclease which is specific for a sequence motif in the genomic DNA thereby producing a second nick in the genomic DNA at the motif sequence,
• c) contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked DNA at the telomere repeat sequence and at the motif sequence;
• and
• d) detecting the length of the telomere by measuring the fluorescence of first fluorescently labeled nucleotide at the telomere repeat location, wherein the fluorescently labeled motif sequences are used as a barcode to identify the chromosome.

A method of detecting the length of an individual telomere, the method comprising:

• a) contacting genomic DNA with a guide RNA having a portion complementary to the telomere repeat sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the telomere repeat sequence;
• b) contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked DNA at the telomere repeat sequence;
• c) contacting the genomic DNA with a second guide RNA having a portion complementary to a sequence in the subtelomeric region of the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the subtelomeric sequence,
• d) contacting the nicked DNA with a polymerase and second fluorescently labeled nucleotide, wherein the second fluorescently labeled nucleotide is incorporated into the nicked DNA at the subtelomeric sequence location;
• and
• e) detecting the length of the telomere by measuring the fluorescence of first fluorescently labeled nucleotide at the telomere repeat location, wherein the fluorescently labeled subtelomeric sequence is used as a barcode to identify the chromosome.

A method of detecting the length of an individual telomere, the method comprising:

• a) contacting genomic DNA with a guide RNA having a portion complementary to the telomere repeat sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the telomere repeat sequence;
• b) contacting the genomic DNA with a second guide RNA having a portion complementary to a sequence in the subtelomeric region of the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the subtelomeric sequence,
• c) contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked DNA at the telomere repeat sequence and at the subtelomeric sequence;
• and
• d) detecting the length of the telomere by measuring the fluorescence of first fluorescently labeled nucleotide at the telomere repeat location, wherein the fluorescently labeled subtelomeric sequence is used as a barcode to identify the chromosome.

A method of detecting an individual haplotype, the method comprising:

• a) contacting genomic DNA with a guide RNA having a portion complementary to a target genomic sequence directly adjacent to a protospacer adjacent motif (PAM), wherein the target genomic sequence or PAM sequence contain different alleles on the maternal or paternal genomic DNA;
• b) contacting gRNA with Cas9 nickase to produce a single-strand nick in the genomic DNA at either maternal or paternal DNA containing the sequence perfectly complementary to the target genomic sequence or PAM sequence, wherein the either maternal or paternal DNA which does not have the perfectly complementary target genomic or PAM sequence is not nicked;
• c) contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotides are incorporated into the nicked DNA at the target genomic or PAM sequence;
• and
• d) detecting the presence of the perfectly matching alleles by detecting the fluorescence of the first fluorescently labeled nucleotide at the target genomic or PAM sequence.

A method of labeling genomic DNA:

• a) contacting genomic DNA with a guide RNA having a portion complementary a first target sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the first target sequence;
• b) contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked DNA at the first target sequence;
• c) contacting the genomic DNA with a second nicking endonuclease which is specific for a sequence motif in the genomic DNA thereby producing a second nick in the genomic DNA at the motif sequence;
• d) contacting the nicked 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 DNA at the motif sequence location.

A method of labeling genomic DNA:

• a) contacting genomic DNA with a guide RNA having a portion complementary a first target sequence in the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the first target sequence;
• b) contacting the nicked DNA with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked DNA at the first target sequence;
• c) contacting the genomic DNA with a second guide RNA having a portion complementary to a second target sequence the genomic DNA and with Cas9 nickase to produce a single-strand break in the genomic DNA at the second target sequence,
• d) contacting the nicked 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 DNA at the second target sequence location.