A versatile method for gene dosage quantification: multiplex PCR and single base extension for copy number and gene-conversion identification of SMN genes

A comparison of the individual genomes within a species demonstrates that structural variation, including copy number variation (CNV), is a major contributor to phenotypic diversity and evolutionary adaptation. CNVs lead to the under/over-expression of a gene, according to the changes in the gene dosage, which account for the development of a number of genomic disorders. Thus, the development of efficient, rapid and accurate CNV screening is of fundamental importance. We report a method that enables the simultaneous determination of the copy numbers of several different targets as well as the discrimination among highly similar/almost identical targets that differ by only one single nucleotide variant, which establishes their copy numbers. The PCR co-amplification and single-base extension technologies are used to identify the copy number of a target sequence relative to a reference sequence of known genomic copy number in a given sample. This efficient and accurate quantification platform was successfully used to quantify the copy numbers of the primary spinal muscular atrophy-determining gene, SMN1, and the disease modifier gene, SMN2. The reliability, low-cost and potential for high-throughput make our method suitable for screening large populations as well as for use as a tool in clinical settings for genetic diagnosis/prognosis.