Multiplex PCR-based real-time invader assay (mPCR-RETINA): a novel SNP-based method for detecting allelic asymmetries within copy number variation regions

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Abstract
We report the development of a real‐time Invader assay combined with multiplex PCR (mPCR‐RETINA), an SNP‐based approach that can measure the allelic ratio in copy number variation (CNV) regions of a genome. RETINA monitors the real‐time fluorescence intensity of each allele during the Invader assay and detects allelic asymmetries caused by genomic duplication/multiplication in heterozygous individuals. By combining mPCR‐RETINA and real‐time quantitative PCR that detects total copy number, we can estimate the copy number of each allele in CNV regions, which should be useful for investigating the functional significance of allele copy number with disease susceptibilities and drug responses. Also, mPCR‐RETINA can efficiently refine the detailed structures of CNV regions. Due to the combination of RETINA with multiplex PCR, mPCR‐RETINA requires a very small amount of genomic DNA for analysis (0.1–0.38 ng/locus). Additionally, mPCR‐RETINA has clear advantages in its simple protocol and target‐specific reaction, even in nonunique regions. We believe mPCR‐RETINA will provide a significant contribution to identifying functional alleles in CNV regions. Hum Mutat 29(1), 182–189, 2008.