Methods of specifically labeling nucleic acids using CRISPR/Cas
A method of detecting the length of an individual telomere is provided. In one embodiment, the method includes contacting genomic DNA with a guide RNA having a portion complementary to a 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. The nicked DNA is contacted with a polymerase and fluorescently labeled nucleotide, wherein the fluorescently labeled nucleotide is incorporated into the nicked DNA at the telomere repeat sequence. The genomic DNA is contacted 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. The nicked DNA is contacted with a polymerase and second fluorescently labeled nucleotide of different color, wherein the second fluorescently labeled nucleotide is incorporated into the nicked DNA at the motif sequence location. The length of the telomere is detected 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.
1. 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 sequence perfectly complementary to the target genomic sequence or PAM sequence by detecting the fluorescence of the first fluorescently labeled nucleotide at the target genomic or PAM sequence
wherein, the haplotype is determined by the detection of said alleles.
2. The method according to claim 1 , further comprising
a) 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;
b) contacting the nicked DNA with a polymerase and second fluorescently labeled nucleotide of different color, wherein the second fluorescently labeled nucleotide is incorporated into the nicked DNA at the motif sequence location, wherein the fluorescently labeled motif sequences are used as a barcode to identify the chromosome.
3. The method according to claim 1 , wherein the genomic DNA is contacted with multiple guide RNAs, each guide RNA having a portion perfectly complementary to a different allele in either the maternal or paternal DNA target genomic or PAM sequence, wherein each allele is detected via fluorescent label, thus providing a haplotype of a portion of the genomic DNA.
4. The method according to claim 1 , wherein the identification of multiple alleles is combined to determine a haplotype.
5. The method according to claim 1 , wherein the fluorescently labeled sequences are used as a barcode to identify the chromosome.
6. The method according to claim 1 , further comprising ligating the labeled DNA with a ligase.
7. The method according to claim 1 , wherein the target sequence is about 20 nucleotides in length, and the guide RNA has exact complementarity to the last 10, 11, or 12 bases of the 20 nucleotide target sequence.
8. The method according to claim 1 , wherein the guide RNA has one or two mismatches in the first 8 nucleotides of the target sequence.
9. The method according to claim 1 , wherein the guide RNA comprises a crRNA and a tracrRNA.
10. The method according to claim 1 , wherein the guide RNA is a single gRNA sequence.
11. The method according to claim 1 , wherein the guide RNA and the Cas9 nickase are contacted with each other to form a complex, prior to contacting with the genomic DNA.
12. The method according to claim 1 , wherein the Cas9 nickase is Cas9 D10A or H840A.