A Sensitive Method for Detecting Variation in Copy Numbers of Duplicated Genes

Gerli Pielberg; Andy E. Day; Graham S. Plastow; Leif Andersson

doi:10.1101/gr.1188203

A Sensitive Method for Detecting Variation in Copy Numbers of Duplicated Genes

¹ Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden
² PIC Group, University of Cambridge, Department of Pathology, Cambridge, UK
³ Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Abstract

Gene duplications are common in the vertebrate genome, and duplicated loci often show a variation in copy number that may have important phenotypic effects. Here we describe a powerful method for quantification of duplicated copies based on pyrosequencing. A reliable quantification was obtained by amplification of the duplication break-point and a corresponding nonduplicated sequence in a competitive PCR assay. A comparison with an independent method for quantification based on the Invader technology revealed an excellent correlation between the two methods. The pyrosequencing-based method was evaluated by analyzing variation in copy number at the duplicated KIT/Dominant white locus in pigs. We were able to distinguish haplotypes at this locus by combining the duplication breakpoint test with a diagnostic test for a functionally important splice mutation in the duplicated gene. An extensive allelic variation, including the presence of a new allele carrying a single KIT copy expected to encode a truncated KIT receptor, was revealed when analyzing white pigs from commercial lines.

Footnotes

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1188203.
↵4 Corresponding author. E-MAIL leif.andersson{at}imbim.uu.se; FAX 46 18 471-4833.
- Accepted June 17, 2003.
- Received January 17, 2003.
Cold Spring Harbor Laboratory Press