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Keywords:DNA microchip, gene synthesis
In a recent publication, Zhang reported a QD based ultrahigh-resolution DNA quantification technique named quantum dot electrophoretic mobility assay (QEMSA). QEMSA exploits the electrohydrodynamic property of streptavidin coated CdSe QDs . Target DNA molecules were tagged with biotin and self assembled onto QD surfaces (Fig. A). The electrophoretic mobility of QDs was precisely modulated by the degree of DNA conjugation. Instead of quantifying based on fluorescent intensities, the amount of target DNA was determined by measuring the relative migration distance of the QD-DNA nanocomplex within a gel (Fig. B). An analytical model based on Poisson-Boltzmann equation predicted the migration distance to be proportional to the logarithm of the DNA to QD ratio N (Fig. C,D). QEMSA enabled accurate quantification down to 1.1-fold (9%) change in quantity. It had been successfully applied to quantify DNA copy number variation of in ovarian cancer cell lines and assess DNA methylation levels of gene promoter.
In addition to environmental conditions, QD emission intensities are strongly influenced by proximal molecules or nanoparticles that QDs interact with. Under many circumstances, the photoluminescence of QDs is drastically quenched through numerous mechanisms. This seemingly undesirable phenomenon can serve as an advantageous feature. For example, quenching mechanism can be designed to act like a molecular switch for fluorescent signals, which would make QDs an ideal homogeneous sensing platform for studying molecular interactions and detecting specific targets.
Gene Synthesis will be available on
Chemiluminescence is another alternative energy source that serves as a FRET donor. For example, Luminol is a chemiluminescent reagent that is activated by oxidants such as H2O2. The reaction requires a catalyst to decompose H2O2 into H2O and O2. The hemin/G-quadruplex horseradish peroxidase (HRP) mimicking catalytic nucleic acids (DNAzymes) were discovered to generate chemiluminescence through catalyzing the oxidation of luminol by H2O2 [, ]. Freeman included aptamer domains into the DNAzyme subunits . One of the aptamer subunits was conjugated to a QD. In the presence of aptamer targets, ATP or Hg2+ in this case, the DNAzyme subunits self assembled into active hemin/G-quadruplex DNAzyme structures and promoted the chemiluminescence resonance energy transfer (CRET) by catalyzing luminol emission. As shown in Figure A, nucleic subunits included domain I and II of the HRP mimicking DNAzyme, as well as domain V and VI of an anti-ATP aptamer. In the absence of ATP, the two subunits are not able to form a stable complex. However, in the presence of ATP, the aptamer domains binds to ATP and the resulting complex leads to the formation of a hemin/G-quadruplex that catalyzes the chemiluminescent reaction and gives rise to CRET. In contrast to FRET, the emission intensities of donors and acceptors increased or decreased concurrently because the amount of energy transferred to the QD was proportional to the chemiluminescent energy available (Fig. B). QD-CRET sensors were also configured to detect specific DNA sequences (Fig. C). A DNA hairpin structure consisting of a few functional domains were conjugated to QDs. The DNAzyme forming domain was blocked in the presence of the hairpin loop. The sequence recognition domain resided in the loop. As the target DNA hybridized to the recognition sequence and opened the hairpin, the DNAzyme forming domain was freed, leading to the self assembly of a hemi/G-quadruplex DNAzyme. DNA hairpins with three different target recognition sequences were conjugated to QD490, QD560 and QD620 respectively to form three QD-CRET DNA probes. Upon hybridization to their respective targets, Hemin and H2O2 were added to induce CRET. The presence of targets was indicated by emission of specific QDs probes through CRET. With the proposed QD-CRET sensor, the authors successfully resolved three targets in a multiplexed format (Fig. D).
Ho developed an imaged based coincidence detection strategy . DNA functionalized QDs hybridized to targets and formed a dual-color labeled sandwich nanoassembly (Fig. A). The presence of targets was identified using colorimetric measurements. Because the physical sizes of QDs were much smaller than the diffraction limited resolution, the co-localized QD pair exhibited the combined color of the two QDs (Fig. B). Meanwhile, unbound QDs retained their original colors, allowing easy differentiation from the target nanocomplexes. Multiplexed detection was accomplished by tagging 6 DNA probes with 3 different QDs. Each target gene hybridized to two probes and resulted in a unique color combination (Fig. C).
) with information about the gene or locus is also included.
For the quantification of geneexpression, researchers have used ß-actin,glyceraldehyde-3-phosphatedehydrogenase (GAPDH), ribosomal RNA (rRNA), or other RNAs as anendogenous control.
Following PCR analysis, thefraction of negative answers is used to generate an absolute answer forthe exact number of target molecules in the sample, without referenceto standards or endogenous controls.
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Accurate multiplex gene synthesis from programmable …
The relative expression ratio of a target gene is computed,based on its real-time PCR efficiencies (E) or a static efficiency of2, and the crossing point (CP) difference (D) of one unknown sample(treatment) versus one control (DCP control - treatment).
Accurate high-throughput gene synthesis using …
The amplification ofChlamydia 23SrRNA allowed for the differentiation of chlamydial species and was morerobust at low target numbers than amplification of the omp1 gene.
Artificial gene synthesis - Wikipedia
Relative quantification is based on the expression levels of a targetgene versus a reference gene and in many experiments is adequate forinvestigating physiological changes in gene expression levels.
Accurate High-throughput Gene Synthesis Using …
As a luminescent probe, QDs outperform traditional organic fluorophores in many aspects due to their excellent optical properties. In addition, a QD can act as a nanoscaffold, thereby providing a structural platform and functional solid substrate for molecule adsorption and interaction. Recently, QDs have been used as active components of complex biosensing platforms which often rely on nonradiative energy transfer between a QD and other organic fluorophores or nanoparticles. Many of these QD-based nano-biosensing techniques enable homogeneous and wash-free assays, thereby eliminating stringent washing steps to greatly simplify assay protocols.
Gene Synthesis Using Programmable Microfluidic ..
Thisaccuracy may only be needed in screening experiments (amount ofmicroorganism in food), to measure the percentage of GMO (geneticmodified organism) in food, to measure the viral load or bacterial loadin immunology and microbiology.
Genetics Real-Time PCR Homepage
The crucial problem in this relative approach is that the mostcommon reference gene transcripts from so-called housekeeping genes,whose mRNA expression can be regulated and whose levels varysignificantly with treatment or between individuals.
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