RESUMEN
Gene transfection vector polyethyleneimine (PEI) was used as a cross-linking agent to crosslink the surface epoxidized magnetic nanoparticles and aggregate them to form a small magnetic bead (MB) with multiple nanoscale bumps on its surface (i.e. the multi-bumpy small magnetic bead, mbsMB). As there is a very low content of non-magnetic components (the cross-linking agent) in the magnetic bead, the mbsMB has an ultrahigh magnetic content of 81.95 % and a smaller particle size of 1.4 µm when compared with the usual medical MB. Such a small MB also has a strong magnetic force allowing it to reach the rapid separating ability of the commonly used larger medical MB which has 8 times its volume. The mbsMB has an obvious pH sensitivity of positive and negative surface charges and the salt-free isolation of DNA has been achieved based on the electrostatic interactions between mbsMB and DNA. This avoids the desalting of the isolated DNA as well as the effects of high salt concentration on its long chain helix structure. Whether in an acidic absorbing medium, an alkalinous desorbing one or a near neutral particle-storing one, the mbsMB will have obvious surface electrostatic charges. There is also its good suspension stability in an aqueous medium which provides a good condition for isolating of DNA suitable for efficiently adsorbing and desorbing. The as-prepared MB has a unique surface structure and some excellent properties, all suitable for adsorbing DNA. In addition, a large amount of commonly used gene transfection vector PEI can be cross-linked and bonded on the surface of mbsMB, whilst still having an excellent DNA-loading ability. In summary, the mbsMB has an ultrahigh capacity of 629.49 mg/g for DNA load.
Asunto(s)
ADN , Polietileneimina , ADN/genética , Concentración de Iones de Hidrógeno , Electricidad Estática , TransfecciónRESUMEN
We have developed a fluorescence double-probe detection system with signal amplification for simple typing and determination of single nucleotide polymorphism (SNP) functional gene based on non-sequence dependence of ExoIII nuclease on dsDNA and rapid separation of magnetic bead. Matched detected gene can cyclically release abundant fluorescence-labeled ssDNA from the probe and the corresponding measured fluorescence signal is amplified up to 6063 times. In this case, the probe cannot release the measured fluorescence signal for the point mutation gene and then the corresponding measured signal is inhibited. According to signal amplification and inhabitation of the probe, we proposed both an accurate genotyping approach with strong specificity and a sensitive determination approach with high selectivity for SNP functional gene. For qualitative genotyping, there are obvious genotype-based differences of measured fluorescence phenotypes among three kinds of the samples of the investigated SNP. The quantitative determinations of its wild-type gene and mutant gene have all a good linearity in the range from 0.5 to 500 pmol/L with the correlation coefficients R2 of 0.9940 and 0.9911, and a high sensitivity with the detection limits of 0.11 and 0.20 pmol/L, respectively. Compared to the usual single-probe detection system, the developed double-probe system can achieve not only accurate genotyping but also the sensitive gene determination. Meanwhile, it is also a simple and reliable method for both quantitative and qualitative analysis of functional gene.