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A sensitive method for detecting variation in copy numbers of duplicated genes
Gerli Pielberg1, Andy E Day, Graham S Plastow
1Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Genome Research
|September 4, 2003
Summary
We developed a pyrosequencing method to accurately quantify gene copy number variations. This method identified new genetic variations in pigs, impacting the KIT gene and potentially leading to a truncated KIT receptor.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Gene duplications are frequent in vertebrates and influence phenotypes through copy number variations.
- Accurate quantification of these variations is crucial for understanding their biological significance.
Purpose of the Study:
- To develop and validate a novel pyrosequencing-based method for quantifying gene copy number variations.
- To apply this method to investigate variations at the KIT/Dominant white locus in pigs.
Main Methods:
- Developed a competitive PCR assay to amplify duplication breakpoints and non-duplicated sequences.
- Quantified gene copies using pyrosequencing.
- Validated the method by comparing it with the Invader technology.
- Analyzed the KIT/Dominant white locus in pigs, combining breakpoint analysis with a splice mutation test.
Main Results:
- The pyrosequencing method provided reliable quantification of gene copy number.
- Excellent correlation was observed between the pyrosequencing and Invader methods.
- Extensive allelic variation was identified at the KIT locus in white pigs.
- A novel allele with a single KIT copy, potentially encoding a truncated KIT receptor, was discovered.
Conclusions:
- The pyrosequencing-based method is a powerful and accurate tool for quantifying gene copy number variations.
- This method revealed significant genetic diversity at the KIT locus in commercial pig lines.
- The identified variations may have important implications for pig coat color and receptor function.