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Copy number variation analysis by ligation-dependent PCR based on magnetic nanoparticles and chemiluminescence.

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 - Theranostics (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1. The State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China;

ABSTRACT
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.

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Effect of different parameters on the relative CL intensity for MNPs-CL system. (a) The concentration of SA modified on the carboxyl MNPs impact on the relative CL intensity; (b) Three different probes hybridized with MNPs impact on the relative CL intensity; (c) Hybridization temperature impact on the relative CL intensity; (d) Number of PCR cycles impact on the relative CL intensity.
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Figure 5: Effect of different parameters on the relative CL intensity for MNPs-CL system. (a) The concentration of SA modified on the carboxyl MNPs impact on the relative CL intensity; (b) Three different probes hybridized with MNPs impact on the relative CL intensity; (c) Hybridization temperature impact on the relative CL intensity; (d) Number of PCR cycles impact on the relative CL intensity.

Mentions: The effects of several reaction parameters were investigated for the detection optimization. The amino groups at surface of the APTS-grafted MNPs were reacted with SA for carboxyl functionalization. The same amount of PCR products from 35 cycles were hybridized with gradiently modified carboxylated probe-MNPs. The values of CL intensity for the MNPs modified with tenfold concentrations of SA (0.1, 0.01, 0.001, 0.0001 and 0.00001M) are shown in Fig. 5a. The CL intensity increased from 0.00001 M to 0.001 M of SA and then decreased under 0.01M SA modification condition. 0.001 M was therefore taken as the optimal concentration for SA modification, and this concentration was chosen for subsequent experiments.


Copy number variation analysis by ligation-dependent PCR based on magnetic nanoparticles and chemiluminescence.

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 - Theranostics (2015)

Effect of different parameters on the relative CL intensity for MNPs-CL system. (a) The concentration of SA modified on the carboxyl MNPs impact on the relative CL intensity; (b) Three different probes hybridized with MNPs impact on the relative CL intensity; (c) Hybridization temperature impact on the relative CL intensity; (d) Number of PCR cycles impact on the relative CL intensity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4265749&req=5

Figure 5: Effect of different parameters on the relative CL intensity for MNPs-CL system. (a) The concentration of SA modified on the carboxyl MNPs impact on the relative CL intensity; (b) Three different probes hybridized with MNPs impact on the relative CL intensity; (c) Hybridization temperature impact on the relative CL intensity; (d) Number of PCR cycles impact on the relative CL intensity.
Mentions: The effects of several reaction parameters were investigated for the detection optimization. The amino groups at surface of the APTS-grafted MNPs were reacted with SA for carboxyl functionalization. The same amount of PCR products from 35 cycles were hybridized with gradiently modified carboxylated probe-MNPs. The values of CL intensity for the MNPs modified with tenfold concentrations of SA (0.1, 0.01, 0.001, 0.0001 and 0.00001M) are shown in Fig. 5a. The CL intensity increased from 0.00001 M to 0.001 M of SA and then decreased under 0.01M SA modification condition. 0.001 M was therefore taken as the optimal concentration for SA modification, and this concentration was chosen for subsequent experiments.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1. The State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China;

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus