Journal
ANALYTICAL BIOCHEMISTRY
Volume 497, Issue -, Pages 95-102Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2015.12.012
Keywords
Allele-specific PCR; SYBR Green-based PCR; Allele-specific copy number
Funding
- Natural Science Foundation of China [81301141]
- Natural Science Foundation of Jiangsu Province of China [BK20131387]
- National Institute of Dental and Craniofacial Research [R15 DE02513801]
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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. (C) 2015 Elsevier Inc. All rights reserved.
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