A novel detection method for heteroduplex DNA using carbodiimide-induced interrupted primer extension

Direct sequencing may be problematic in demonstrating mutations where inherited disease results from multiple different heterozygous variants in large genes. We describe here a novel mutation screening method based on the ability of carbodiimide to bind mismatched DNA and interrupt primer extension thereby identifying both a heterozygous variant and its location. This assay detected all four classes of DNA mismatch in 550 bp engineered plasmid fragments and in two dominantly inherited renal diseases. In patients with thin basement membrane nephropathy, the method demonstrated multiple variants within a single amplicon including some close to the primer binding site. This method also detected a complex mutation in medullary cystic kidney disease type 2 (c.278-289 del/insCCGGCTCCT) as multiple termination events and, furthermore, correctly identified five affected and 28 unaffected family members. Carbodiimide-induced interrupted primer extension identifies heterozygous variants in large or multiexonic genes, where the variants differ in each family, their locations are unknown, and even if there are multiple known non-pathogenic variants within the same amplicon. This assay incorporates a “universal” protocol that detects all types of mutations without the need for further optimization, and potentially detects mutations where the proportion of heteroduplex is less than 50%.