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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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Related in: MedlinePlus

Diagrammatic sketch of corresponding positions for three hybridization probes: (a) 5'NH2-(T)15-probe; (b) 3'NH2-(T)15-probe; (c) 3'NH2-probe.
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Figure 3: Diagrammatic sketch of corresponding positions for three hybridization probes: (a) 5'NH2-(T)15-probe; (b) 3'NH2-(T)15-probe; (c) 3'NH2-probe.

Mentions: The optimization of CL system consisted of probe optimization, PCR cycle number, and hybridization temperature. Three probes immobilized onto the carboxylated MNPs were designed into following groups: 5'NH2-(T)15-probe (Fig. 3a), 3'NH2-(T)15-probe (Fig. 3b) and 3'NH2-probe (Fig. 3c). The amino ends were immobilized at the surface of the carboxylated MNPs. After ligation, the products were amplified by PCR with 20, 25, 30, 35 and 40 cycles, respectively. The PCR products were then hybridized with probe-MNPs at following gradient temperatures: 50℃, 52℃, 54℃, 56℃, 58℃, 60℃. After hybridization and CL detection, the most suitable and efficient probe, PCR cycle number and hybridization temperature were determined.


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)

Diagrammatic sketch of corresponding positions for three hybridization probes: (a) 5'NH2-(T)15-probe; (b) 3'NH2-(T)15-probe; (c) 3'NH2-probe.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Diagrammatic sketch of corresponding positions for three hybridization probes: (a) 5'NH2-(T)15-probe; (b) 3'NH2-(T)15-probe; (c) 3'NH2-probe.
Mentions: The optimization of CL system consisted of probe optimization, PCR cycle number, and hybridization temperature. Three probes immobilized onto the carboxylated MNPs were designed into following groups: 5'NH2-(T)15-probe (Fig. 3a), 3'NH2-(T)15-probe (Fig. 3b) and 3'NH2-probe (Fig. 3c). The amino ends were immobilized at the surface of the carboxylated MNPs. After ligation, the products were amplified by PCR with 20, 25, 30, 35 and 40 cycles, respectively. The PCR products were then hybridized with probe-MNPs at following gradient temperatures: 50℃, 52℃, 54℃, 56℃, 58℃, 60℃. After hybridization and CL detection, the most suitable and efficient probe, PCR cycle number and hybridization temperature were determined.

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