Probe-free allele-specific copy number detection and analysis of tumors

• Published in: Analytical biochemistry Vol. 497; pp. 95 - 102
• Main Authors: Zhu, Ailin, Guan, Xiaowei, Gu, Xinbin, Xie, Guiqin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2016
• Subjects: Allele-specific copy number; Allele-specific PCR; Alleles; Animals; carcinogenesis; Cell Line, Tumor; DNA; DNA - genetics; DNA Copy Number Variations; Fluorescent Dyes - chemistry; Gene Dosage; genotyping; Genotyping Techniques - methods; Humans; medicine; melting; Mice; neoplasms; Neoplasms - genetics; Nucleic Acid Denaturation; Organic Chemicals - chemistry; Polymorphism, Single Nucleotide; PTEN Phosphohydrolase - genetics; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; single nucleotide polymorphism; SYBR Green-based PCR; ddPCR; Allele-specific PCR; Allele-specific copy number; ANOVA; Tm; SYBR Green-based PCR; PCR
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2015.12.012

Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping.