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

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.

CYP2D6 Genotyping for Functional-Gene Dosage Analysis by Allele Copy Number Detection

“…Recently, we developed a multiplex PCR-based real-time Invader assay (mPCR-RETINA) that reveals the allele ratio at each locus within CNV regions by detecting allelic asymmetries (18 ). By combining the allele ratio data with the data for total gene copy number obtained by real-time quantitative PCR (qPCR), we were able to measure the allele copy number at each locus (18 ).…”

“…By combining the allele ratio data with the data for total gene copy number obtained by real-time quantitative PCR (qPCR), we were able to measure the allele copy number at each locus (18 ). We also developed CNVphaser software that can estimate both the haplotype frequency in a population and the diplotype of each individual within CNV regions (19 ).…”

“…Copy number was calculated by the ⌬⌬Ct method, where Ct is the threshold cycle (18,(21)(22)(23). The PCR efficiency of each assay was estimated from the calibration curves of a sample (NA18995) serially diluted from 40 ng to 2.5 ng of genomic DNA.…”

“…Details for determining the allele ratio by RETINA have previously been described (18 ). For the RETINA assay, we first amplified an entire CYP2D6 gene region via a triplex PCR with the previously reported primers (9,15 ).…”

“…The allele ratio at each locus was determined in each sample by analysis of allelic discrimination plots from calibration curves of the allele ratio (see Fig. 3 in the online Data Supplement) (18 ).…”