Robust dosage-PCR for detection of heterozygous chromosomal deletions

Robust dosage-PCR (RD-PCR) was developed to detect heterozygous large deletions, an important class of mutations missed by conventional PCR strategies. PCR-based methods are available for distinguishing between the dosage of one or two template copies, but general application is limited by the laborious nature of the method and/or the optimization required for each new set of gene exons to be analyzed. RD-PCR depends on a combination of (i) co-amplification of an autosomal and an X-chromosomal segment so that internal dosage controls are available for any segment to be analyzed and (h) a robust primer design that includes a 5' tail and a 3' sequence-specific region in the PCR protocol. The ratio of yields (ROY) of the target to the internal control segment is directly proportional to the ratio of the two input templates over a wide range (at least 1:1 to 1:258 with a correlation coefficient of 0.99). The ROY is not dependent on the amount of genomic DNA or the number of cycles of amplification under typical conditions. RD-PCR eliminates errors in the preparation and manipulation steps by using an internal dosage control. A blinded analysis of gene dosage was performed to detect deletions of the human factor IX gene with 100% accuracy. Prospective analyses demonstrate that exons and flanking splice junctions can be analyzed for gene dosage with minimal optimization.