A large-scale validation of dosage analysis by robust dosage-polymerase chain reaction

Epidemiological and clinical diagnostic assays benefit from accurate detection of deletions and duplications commonly missed by the conventional strategy of polymerase chain reaction (PCR) amplification and sequencing of individual exons. Robust dosage-PCR (RD-PCR) is a quantitative duplex PCR method that coamplifies a target template and an endogenous internal control (an autosomal and an X-chromosomal segment) for detection of these mutations. In this study, 110 consecutive RD-PCR assays were developed and validated. The average linear regression coefficient between template copy number and product yield and the average coefficient of determination for linear correlation, R2, were very high: 0.95 and 0.98, respectively. The accuracy of RD-PCR revealed somatic mosaicism for a deletion in the factor 9 gene. Advantages of RD-PCR include (1) high accuracy and consistency, (2) easy calibration of linearity using male and female samples, (3) use of an endogenous internal dosage control to eliminate preparation and manipulation errors, and (4) detection of gene dosage over a wide dynamic range. Deletions and duplications can be easily detected (a 2× decrease or a 1.5× increase in gene dosage). Thus, RD-PCR is a general and accurate method for detecting changes in gene dosage.