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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

The gel electrophoresis results of ligation-dependent PCR for six detected genes. Sample 1 was male, sample 2 was female and CK was the blank control performed under the same reaction compositions without DNA template.
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Figure 7: The gel electrophoresis results of ligation-dependent PCR for six detected genes. Sample 1 was male, sample 2 was female and CK was the blank control performed under the same reaction compositions without DNA template.

Mentions: To validate the methodology for the present new system, six genes were selected for copy number analysis in human genome (Table 1). The amplification products were visualized by agarose gel electrophoresis and the specificity of the designed probes was analyzed (Fig. 7). It was shown that the target bands for six gene loci were yielded at the range of 110 bp to 120 bp, while there were no nonspecific bands in the blank control. As shown in Fig. 7b, the positive bands for SRY and DAZ1 only appeared in male (sample 1) and no corresponding amplification was found in female (sample 2), owing to these two genes only exist on the Y-chromosome, which could also be used for gender determination. These results suggested that the designed probes for target genes were able to amplify all loci presented in the samples, and represented high specificity without cross-reactions. The brightness of the target bands showed the high amplification efficiency of universal primers, laying the foundations for the subsequence experiments of copy number quantification.


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)

The gel electrophoresis results of ligation-dependent PCR for six detected genes. Sample 1 was male, sample 2 was female and CK was the blank control performed under the same reaction compositions without DNA template.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: The gel electrophoresis results of ligation-dependent PCR for six detected genes. Sample 1 was male, sample 2 was female and CK was the blank control performed under the same reaction compositions without DNA template.
Mentions: To validate the methodology for the present new system, six genes were selected for copy number analysis in human genome (Table 1). The amplification products were visualized by agarose gel electrophoresis and the specificity of the designed probes was analyzed (Fig. 7). It was shown that the target bands for six gene loci were yielded at the range of 110 bp to 120 bp, while there were no nonspecific bands in the blank control. As shown in Fig. 7b, the positive bands for SRY and DAZ1 only appeared in male (sample 1) and no corresponding amplification was found in female (sample 2), owing to these two genes only exist on the Y-chromosome, which could also be used for gender determination. These results suggested that the designed probes for target genes were able to amplify all loci presented in the samples, and represented high specificity without cross-reactions. The brightness of the target bands showed the high amplification efficiency of universal primers, laying the foundations for the subsequence experiments of copy number quantification.

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