A TaqMan(R) real time PCR based method for studying copy number variations

摘要

Copy number variation (CNV), a form of genetic variations, is usually defined as a segment of genomic DNA longer than 1kb* involving in deletions or insertions. Copy number variations are extensively present in the gehomes of human and non-human species.Some of them are directly linked to biological functions and may contribute to genetic disorders and diseases. Copy number variations are important for understanding genetic diversity and genetic disorders, developing genetic diagnostic tests, and providing insights on evolution.We developed quantitative TaqMan(R) real time PCR based assays (TaqMan(R) Copy Number Assays) for detecting and measuring CNV regions. TaqMan(R) Copy Number Assays are an assay duplex: a TaqMan(R) Copy Number Assay and a TaqMan(R) Copy Number Reference Assay. TaqMan(R) Copy Number Assay is a FAMTM dye-based assay designed to detect a target-of-interest on genome. TaqMan(R) Copy Number Reference Assay is a VIC(R) dye-based assay designed for a reference sequence known to be present in two copies in a diploid genome.Each assay reaction is performed on an Applied Biosystems Real-Time PCR instrument with an assay duplex and a genomic DNA sample. The sample's copy number is determined by relative quantity of the target-of -interest to a calibrator sample with a known copy number. TaqMan(R)Copy Number Assays were designed in-silico against predefined human sequence targets using our proprietary algorithms. We then randomly selected assays for lab test based on their genomic penalties, thermodynamic properties, oligo interactions among primers and probes, interactions between target and reference assays. The experimental results provided feedback to optimize assay design algorithms and fine tune assay selection criteria. Through this iterative assay development process, we have designed and selected over 670K TaqMan(R) Copy Number Assays targeting known gene and CNV regions for public launch in the early 2009.Finally, we developed the software CopyCaller(TM) to analyze the results of TaqMan(R) Copy Number Assay experiments. The software allows users to make copy number calls, remove outliers, adjust analysis parameters, compare the data from multiple experiments, and visualize the data in graphic and tabular formats. The software is expected to be available for free public access in the early 2009.