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INTRODUCTION  The accurate measurement of the copy number (CN) for an allele is often desired. We have developed a simple pyrosequencing-based method, reference query pyrosequencing (RQPS), to determine the CN of any allele in any genome by taking advantage of the fact that pyrosequencing can accurately measure the molar ratio of DNA fragments in a mixture that differ by a single nucleotide. The method involves the preparation of an RQPS probe, which contains two linked DNA fragments that match a reference allele with a known CN and a query allele with an unknown CN. In each fragment, a single nucleotide variation (SNV) is engineered to differentiate it from its genomic counterparts when the probe is mixed with genomic DNA. The ratios of the two pairs of fragments (probe reference vs. genomic reference and probe query vs. genomic query) in the mixture reflect the ratio between the probe and the genomic DNA in a CN-dependent manner. Pyrosequencing can be used to quantify these ratios and thus determine the CN of the query allele. This method could be used to measure the CN of any transgene, differentiate homozygotes from heterozygotes, detect the copy number variation (CNV) of endogenous genes, and screen embryonic stem (ES) cells targeted with bacterial artificial chromosome (BAC) vectors that are not compatible with standard screening methods.

Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS)