Abstract 4923: Detecting copy number variation using dNTP limited PCR and high-resolution melting
Authors: Zhou Luming , Palais Bobert , Wittwer Carl 
Published: August 2015

Background: Copy number variation (CNV) is a common type of genetic variation. About 13% of genes in the human genome have variation in copy number. Copy number alterations (CNAs), somatic changes to chromosome structure that result in gain or loss in copies of sections of DNA, are very common in cancer and associated with particular cancer types. In some cases such as EGFR and HER2, the copy number alterations have led to the identification of cancer-causing genes and suggested specific therapeutic approaches.

Materials and Methods: Target and reference regions of genomic DNA were amplified together using a variety of amplification limiting PCR reagent and cycling protocols. Ratios of reference and copy number variants were maintained by limiting quantities of dNTPs and allowing PCR to plateau. Suitable fragments having single melting domains, well-separated Tms, and no common homologs were designed using uMelt melting curve prediction software. After PCR, high-resolution melting (fluorescence vs. temperature) data was acquired from double stranded DNA dye LCGreen Plus present in the reaction. MeltWizard 6 software was used to remove background, equalize negative derivative melting peaks corresponding to the reference segment, and identify and quantify samples with different CNV ratios from the amplitude of their target melting peaks. The protocols were optimized on samples with known ratios.

Results: All proposed methods of limiting amplification (cycle number, dNTPs, Taq polymerase) were found to be capable of preserving initial copy ratios sufficiently to identify all common human copy number variation ratios when used in conjunction with high-resolution melting peak reference normalization. CNV ratios far beyond this range, as small as 2 : 2.125 (5.88%), were detectable using the most robust, simplest, and highest resolution method of limiting dNTPs at an optimal 3.25 uM. Duplex PCR of reference CFTR and target EGFR genes were used to detect the copy number variation. Copy number of EGFR in 8 NSCNC lung cancer and 41 colon cancer samples were detected with 6.25 uM dNTP. In 7 out of 8 of lung cancer and 7 out of 41 colon cancer samples, the copy number of EGFR is higher than normal copy 2. The Tm of the reference peak may be lower (duplication) or higher (deletion) than that of the CNV target without affecting the results.

Conclusion: Copy number variation can be quantified using reaction limited multiplex PCR followed by high-resolution melting analysis. This simple closed-tube method is fast, economical, more accurate and less susceptible to contamination than other methods. Assays are easy to design, limiting dNTPs is simple, and the results are stable and reliable. After PCR, only 5 to 10 minutes of melting and analysis are required to obtain results.

Citation Format: Luming Zhou, Bobert Palais, Carl Wittwer. Detecting copy number variation using dNTP limited PCR and high-resolution melting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4923. doi:10.1158/1538-7445.AM2015-4923