title: Probe-free allele-specific copy number detection and analysis of tumors.
abstract: 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.
authors.affiliation: Department of Physiology, Nanjing Medical University, Nanjing 210029, China; Howard University, Washington, DC 20059, USA.
authors.forename: Ailin
authors.initials: A
authors.lastname: Zhu
authors.affiliation: Department of Physiology, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Guan
authors.affiliation: Howard University, Washington, DC 20059, USA.
authors.forename: Xinbin
authors.initials: X
authors.lastname: Gu
authors.affiliation: Department of Physiology, Nanjing Medical University, Nanjing 210029, China. Electronic address: serene_x@163.com.
authors.forename: Guiqin
authors.initials: G
authors.lastname: Xie
mesh_terms: D000483:Alleles; D000818:Animals; D052160:Benzothiazoles; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics; D056915:DNA Copy Number Variations; D003959:Diamines; D005456:Fluorescent Dyes / Q000737:chemistry; D018628:Gene Dosage; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals / Q000737:chemistry; D051059:PTEN Phosphohydrolase / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D004247:DNA; D051059:PTEN Phosphohydrolase
keywords: Allele-specific PCR; Allele-specific copy number; SYBR Green-based PCR