Drexel University

Temple University

The Wistar Institute

Jennifer McCaffrey

Ming H Xiao

Wenhui Hu

Harold C Riethman

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.

Methods of specifically labeling nucleic acids using CRISPR/Cas

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.

Growing community of inventors

Collegeville, PA, United States of America

Jennifer McCaffrey