Digital assay for telomere length

摘要

A method of characterizing telomere length. A template may be synthesized in a bulk phase reaction mixture containing genomic DNA. Partitions may be formed after synthesizing the template in the bulk phase reaction mixture, with only a subset of the partitions containing at least one copy of the template. At least a region of the template may be amplified in partitions. Amplification data may be collected from partitions. A measure of telomere length may be determined for the genomic DNA based on the amplification data. In some embodiments, the bulk phase reaction mixture may be thermally cycled for at least two cycles, and heated to a denaturation temperature and cooled to an annealing temperature in each cycle.

主权项

1. A method of characterizing telomere length, the method comprising:
synthesizing a template with a first primer and a second primer in a bulk phase reaction mixture containing genomic DNA, the template representing a repetitive telomeric sequence present in the genomic DNA and containing a binding site introduced by one of the primers, wherein the one primer includes a 3′ portion that binds a repetitive telomeric sequence in the genomic DNA and also includes a 5′ portion that is not required for binding to the repetitive telomeric sequence, and wherein the 5′ portion of the one primer introduces the binding site; forming partitions after synthesizing the template in the bulk phase reaction mixture, with only a subset of the partitions containing at least one copy of the template; amplifying at least a region of the template in partitions to form an amplicon in the presence of a probe that specifically binds to a region of the amplicon that corresponds to the binding site; collecting amplification data from the probe in partitions; and determining a measure of telomere length for the genomic DNA based on the amplification data.