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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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(a) The ligation-dependent PCR products amplified by 35 cycles, and lane 1 represents the blank control and lane 2 represents the product. (b) The curve of CL detection for PCR products, and the blank control group was performed under the same reaction conditions without template.
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Figure 4: (a) The ligation-dependent PCR products amplified by 35 cycles, and lane 1 represents the blank control and lane 2 represents the product. (b) The curve of CL detection for PCR products, and the blank control group was performed under the same reaction conditions without template.

Mentions: As illustrated in Fig.1, the two oligonucleotides were hybridized to the DNA template and ligated by the ligase enzyme to form a ligated full-length sequence that could be amplified with a pair of universal primers. The PCR product was analyzed with agarose gel electrophoresis. Result showed that there was an amplification target band at the 120 bp and there was no amplification bands in the blank (Fig. 4a), demonstrating that the two probes had been ligated together and amplified with the ends of universal primers. It was thus suggested that the designed ligation probes and universal primers were suitable for the ligation-dependent PCR system due to their good specificity. The CL detection was performed after the dUTP-labeled PCR products were captured by the probe-MNPs. The CL intensity profile was related with the reaction time of STV-AP with AMPPD. As shown in Fig. 4b, the CL signal rose rapidly and became stable at 50 min. The maximum CL value of PCR products was remarkably higher than that of the blank control group. It was therefore suggested that this CL detection system, consisting of ALP and AMPPD, showed a stable light signal and was sensitive to the ligation-dependent PCR product which was labeled with dUTP.


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)

(a) The ligation-dependent PCR products amplified by 35 cycles, and lane 1 represents the blank control and lane 2 represents the product. (b) The curve of CL detection for PCR products, and the blank control group was performed under the same reaction conditions without template.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: (a) The ligation-dependent PCR products amplified by 35 cycles, and lane 1 represents the blank control and lane 2 represents the product. (b) The curve of CL detection for PCR products, and the blank control group was performed under the same reaction conditions without template.
Mentions: As illustrated in Fig.1, the two oligonucleotides were hybridized to the DNA template and ligated by the ligase enzyme to form a ligated full-length sequence that could be amplified with a pair of universal primers. The PCR product was analyzed with agarose gel electrophoresis. Result showed that there was an amplification target band at the 120 bp and there was no amplification bands in the blank (Fig. 4a), demonstrating that the two probes had been ligated together and amplified with the ends of universal primers. It was thus suggested that the designed ligation probes and universal primers were suitable for the ligation-dependent PCR system due to their good specificity. The CL detection was performed after the dUTP-labeled PCR products were captured by the probe-MNPs. The CL intensity profile was related with the reaction time of STV-AP with AMPPD. As shown in Fig. 4b, the CL signal rose rapidly and became stable at 50 min. The maximum CL value of PCR products was remarkably higher than that of the blank control group. It was therefore suggested that this CL detection system, consisting of ALP and AMPPD, showed a stable light signal and was sensitive to the ligation-dependent PCR product which was labeled with dUTP.

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