Theranostics 2015; 5(1):71-85. doi:10.7150/thno.10117
Copy Number Variation Analysis by Ligation-Dependent PCR Based on Magnetic Nanoparticles and Chemiluminescence
1. The State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China;
2. Nanjing Maternity and Child Health Care Hospital, Nanjing 210029, China;
3. Nanjing Medical University, Nanjing 210029, China;
4. Hunan Key Laboratory of Green Packaging and Application of Biological Nanotechnology, Hunan University of Technology, Zhuzhou 412007, P. R. China.
Liu M, Hu P, Zhang G, Zeng Y, Yang H, Fan J, Jin L, Liu H, Deng Y, Li S, Zeng X, Elingarami S, He N. Copy Number Variation Analysis by Ligation-Dependent PCR Based on Magnetic Nanoparticles and Chemiluminescence. Theranostics 2015; 5(1):71-85. doi:10.7150/thno.10117. Available from http://www.thno.org/v05p0071.htm
A novel system for copy number variation (CNV) analysis was developed in the present study using a combination of magnetic separation and chemiluminescence (CL) detection technique. The amino-modified probes were firstly immobilized onto carboxylated magnetic nanoparticles (MNPs) and then hybridized with biotin-dUTP products, followed by amplification with ligation-dependent polymerase chain reaction (PCR). After streptavidin-modified alkaline phosphatase (STV-AP) bonding and magnetic separation, the CL signals were then detected. Results showed that the quantification of PCR products could be reflected by CL signal values. Under optimum conditions, the CL system was characterized for quantitative analysis and the CL intensity exhibited a linear correlation with logarithm of the target concentration. To validate the methodology, copy numbers of six genes from the human genome were detected. To compare the detection accuracy, multiplex ligation-dependent probe amplification (MLPA) and MNPs-CL detection were performed. Overall, there were two discrepancies by MLPA analysis, while only one by MNPs-CL detection. This research demonstrated that the novel MNPs-CL system is a useful analytical tool which shows simple, sensitive, and specific characters which are suitable for CNV analysis. Moreover, this system should be improved further and its application in the genome variation detection of various diseases is currently under further investigation.
Keywords: Copy number variation, Ligation-dependent PCR, Magnetic nanoparticles, Chemiluminescence.