Phasing DNA Markers Using Digital PCR

: Besides quantifying the absolute number of copies of known DNA targets, digital PCR can also be used to assess whether two nonpolymorphic gene sequences or two heterozygous markers reside on the same DNA molecule (i.e., are physically linked). Some useful linkage applications include: phasing variants to define a haplotype; genotyping of inversions; determining the presence of multimarker pathogenic bacteria in a metagenomic sample; and assessing DNA integrity. This chapter describes an efficient and cost-effective method for analyzing linkage of any two genetic sequences up to at least 200 Kb apart, including phasing of heterozygous markers such as that which occur abundantly in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.