Research Of Nucleic Acid Quantification And Copy Number Variation Analysis Based On Magnetic Nanoparticles And Chemiluminescence

Copy number variation (CNV) is a structurally variant region ranging from 1 kilobases (kb) to megabases (Mb) in size. It presents at variable copy number in comparison with a reference genome, including deletions, insertions, duplications and complex multi-site variants. CNVs are widely distributed in the genome, influencing gene expression, phenotypic variation and altering gene dosage, and can cause various complex diseases. The study of CNVs can help us explore the molecular mechanism of genetic diseases and find molecular markers for genetic diagnosis and prenatal diagnosis, which can provide the scientific basis for the molecular diagnosis and treatment of diseases. With the rapid development of nanotechnology in life science, nanomaterials, especially magnetic nanoparticles (MNPs), have been widely used in biomedical scopes owing to their excellent separation and adsorption capacity, good biocompatibility and easy operation in automated work-stations. In addition, chemiluminescence (CL) detection is regarded as an effective method for biological detection because of its extremely high sensitivity, specificity and simple instruments. In this study, combining the merits of MNPs and CL detection, we developed a new system for nucleic acid quantification and CNV analysis quickly, sensitively and practicably. The main contents are as follows:1. Preparation of magnetic composite particlesFe3O4 nanoparticles were prepared with uniform shape and good dispersion by the solvothermal method, and then coated with silica to form Fe3O4/SiO2 magnetic composite particles. Characterized by transmission electron microscopy (TEM) and scanning electron microscope (SEM), Fe3O4/SiO2 MNPs were spherical solid nanoparticles with core-shell structures and had a diameter of 450 nm. After the modification of functional groups on the surface of Fe3O4/SiO2 MNPs, the carboxylated MNPs were prepared for the immobilization of capture probes.2. A preliminary study for nucleic acid quantification by primer extension based on magnetic nanoparticles and chemiluminescenceThe origination of copy number variation is the alteration of gene dosage, which indicates CNV detection and analysis should be based on nucleic acid quantification. In this study, a new method for nucleic acid quantification was preliminarily explored by a combination of magnetic separation and CL detection technique. The artificial sequences GAPDH, GSTT1 and GSTM1 were used as the target genes in such method. The primer extended with biotin-dUTP and then hybridized with capture probes which were immobilized onto carboxylated MNPs. After bonding with streptavidin-modified alkaline phosphatase (STV-AP) and magnetic separation, 3-(2’-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) was added, the CL signals were then detected. Preliminary results showed that the extension product could be reflected by CL signal values with a good specificity. The conditions of MNPs modification, hybridization and CL detection were optimized, including the concentration of succinic anhydride (SA) for carboxyl modification, concentration of amino-modified probes, MNPs amounts and hybridization temperature. Under optimum conditions, the MNPs-CL system was characterized for quantitative analysis and the CL intensity exhibited a linear correlation with the target concentration ranging from 0.625 μM to 10 μM, which showed that this system could be used in quantitative analysis for nucleic acids in an appropriate concentration range. However, a high detection limit isn’t suitable for quantitative anlaysis in genomic DNA samples. The sensitivity of this system should be improved further to widen the linear range of CL detection.3. Research of a new system for nucleic acid quantification by ligation-dependent PCR based on MNPs and CL detectionTo realize signal amplifications for quantitative detection in genomic DNA, a novel system was developed by ligation-dependent PCR based on MNPs and CL detection. Two oligonucleotides were hybridized to the artificial template (GAPDH) 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 products with the base insertion of biotin-dUTP were captured by amino-modified probes immobilized on carboxyl MNPs (probe-MNPs). After STV-AP bonding and magnetic separation, the CL signals were detected by the reaction of STV-AP with AMPPD. The effects of several reaction parameters were investigated for the detection optimization, including the MNPs amounts, carboxyl concentration to amino-MNPs, structure and concentration of specific amino-probes, hybridization temperature and binding capacity of probe-MNPs. Under optimum conditions, the MNPs-CL system was characterized for quantitative analysis and the CL intensity exhibited a linear correlation with logarithm of the target concentration ranging from 0.02 pM to 200 pM (R2=0.987). It was proved that the novel system provided good specificity and sensitivity for DNA detection, which offered the technological foundation for quantitative detection and CNV analysis in genimic DNA.4. Primary CNV anlaysis by ligation-dependent PCR based on MNPs and CL detectionThe techniques of MNPs and chemiluminescence were employed to fabricate the DNA biosensors in some previous researches, but the reported target sequences were almost small fragments of artificial single stranded DNA, and detecting double stranded and longer DNA using the technology mentioned above has never been reported previously. To validate the methodology of the novel quantification system by liagtion-dependent PCR based on MNPs and CL detection, CNV detection was performed in genomic DNA. The preliminary result of copy number analysis for artificial genes inserted in the plasmid DNA showed that the MNPs-CL system had a good specificity in CNV detection in genomic DNA. Copy numbers of six genes from the human genome were detected subsequently in 40 samples including male, female, normal and trisomy 21. To compare the detection accuracy, multiplex ligation-dependent probe amplification (MLPA) and MNPs-CL detection were performed simultaneously. Overall, there were two discrepancies by MLPA analysis, while only one by MNPs-CL detection. This research demonstrated that the sensitivity and specificity of the novel MNPs-CL system were equivalent to that from the MLPA system, which was suitable for CNV analysis in human genome.5. Exploration for nucleic acid quantification based on gold-coated magnetic nanocomposites and fluorescenceIn our previous research, we explored some other approaches for nucleic acid quantification based on gold-coated magnetic nanocomposites (GMNPs) and fluorescence. Two novel systems were developed by in situ primer extension (ISPE) and ligation-dependent PCR (LD-PCR), respectively. The SA-modified Fe3O4@SiO2@Au GMNPs were used as preferred carriers and the artificial sequence was used as the target gene in such methods. Some experimental parameters had been optimized, including the GMNPs amounts, concentration of fluorescence probes and hybridization temperature in ISPE system, and the component of buffers in LD-PCR system. Under optimum conditions, the two systems were characterized for quantitative analysis. In ISPE system, the fluorescence intensity exhibited a linear correlation with the target concentration ranging from 0.0125μM to 0.1 μM, and seems not good for quantitative anlaysis in genomic DNA samples due to its low detection limit. In LD-PCR system, the fluorescence intensity did not show an ideal linear correlation with the logarithm of the target concentration, which suggested that this system could not be used in quantitative analysis for nucleic acids at its present form.