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Copyright © 2015 American Scientific Publishers
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Article
Journal of
Nanoscience and Nanotechnology
Vol. 15, 1123–1129, 2015
www.aspbs.com/jnn
Fluorescent Quantification of DNA Based on Core–Shell
Fe
3
O
4
@SiO
2
@Au Nanocomposites and Multiplex
Ligation-Dependent Probe Amplification
Jing Fan
1 2†
, Haowen Yang
1 3†
, Ming Liu
1†
, Dan Wu
4
, Hongrong Jiang
1
, Xin Zeng
1
,
Sauli Elingarami
1
, Zhiyang Li
1
, Song Li
5
, Hongna Liu
1
, and Nongyue He
1 5 ∗
1
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China
2
Clinical Laboratory, Nanxishan Hospital of Guangxi Zhuang Nationality Autonomous Region, Guilin 541002, China
3
School of Animal Science and Technology, Guangxi University, Nanning 530004, China
4
Teaching and Experiment Center of Basic Medicine of Guilin Medical University, Guilin 541002, China
5
Hunan Key Laboratory of Green Packaging and Biological Nanotechnology, Hunan University of Technology,
Zhuzhou 412008, China
In this research, a novel method for relative fluorescent quantification of DNA based
on Fe
3
O
4
@SiO
2
@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation-
dependent probe amplification (MLPA) has been developed. With the help of self-assembly,
seed-mediated growth and chemical reduction method, core–shell Fe
3
O
4
@SiO
2
@Au GMNPs were
synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which
can capture the biotinylated probes. Then we designed MLPA probes which were tagged with
biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the
thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with
SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed
our method can be used for the relative quantitative analysis of the target DNA in the concentration
range of 0004∼05 M.
Keywords: GMNPs, MLPA, Fluorescence Detection, Alzheimer’s Disease, Copy Number
Variations.
1. INTRODUCTION
Gold-coated magnetic nanocomposites (GMNPs) have
been widely applied in separation, purification and detec-
tion of biomolecules, and also in the fields of targeted
drug therapy and functional magnetic resonance imag-
ing (FMRI).
1–8
Compared with other MNPs, the GMNPs
reveal a lower fluorescent background, giving a higher
signal-to-noise ratio and resolution in the application of
detecting the fluorescent markers.
9–11
Copy number variations (CNVs) is a type of recently
discovered genetic markers which are often long DNA
nucleic acid fragments (≥1 kb).
12–15
The genome coverage
of CNVs is more than single nucleotide polymorphisms
∗
Author to whom correspondence should be addressed.
†
These three authors contributed equally to this work.
(SNPs)—another common type of genetic markers. Recent
studies have shown that the human susceptibility to vari-
ous diseases may be associated with CNVs, as the autoso-
mal dominant early-onset Alzheimer disease (ADEOAD),
schizophrenia, autism, etc. The key points of the CNVs
researches are the quantitation and orientation for the
CNV sequences. The multiplex ligation-dependent probe
amplification (MLPA) technology which combines the
specificity of ligase chain reaction (LCR) and the expo-
nential amplification of polymerase chain reaction (PCR),
has become one of the commonly used detection methods
for CNVs.
16–20
In this research, based on the sequence of the human
beta amyloid precursor protein (APP) gene which was
reported on the GenBank,
21
we designed a pair of Ligase
detection reaction (LDR) probes which were modified by
J. Nanosci. Nanotechnol. 2015, Vol. 15, No. 2 1533-4880/2015/15/1123/007 doi:10.1166/jnn.2015.8910 1123