Controllable extension of hairpin-structured flaps to allow low-background cascade invasive reaction for a sensitive DNA logic sensor for mutation detection.

A DNA logic sensor was constructed for gene mutation analysis based on a novel signal amplification cascade by controllably extending a hairpin-structured flap to bridge two invasive reactions. The detection limit was as low as 0.07 fM, and the analytical specificity is high enough to unambiguously pick up 0.02% mutants from a large amount of wild-type DNA. Gene mutations related to the personalized medicine of gefitinib, a typical tyrosine kinase inhibitor, were analyzed by the DNA logic sensor with only a 15 minute response time. Successful assay of tissue samples and cell-free plasma DNA indicates that the new concept we proposed here could benefit clinicians for straightforward prescription of a mutation-targeted drug.

Progress in multiplex loop-mediated isothermal amplification technology.

Loop-mediated isothermal amplification (LAMP) has been widely applied in nucleic acid diagnostics due to its high sensitivity and specificity, high speed and low requirement of equipment. In order to fully leverage these merits, achieve high efficiency and reliability in diagnostics, and expand the applicable fields while keeping low reagent cost, multiplex LAMP technology has been extensively explored in recent years. Common methods for LAMP products detection are mostly based on the double-stranded DNA amplicons or byproducts from the polymerization reaction, so they can only identify the occurrence of amplification reaction but not the origins or specificity of the products. To achieve specific LAMP products detection, researchers developed various multiplex methods by improving the conventional LAMP technology or coupling LAMP with other assays. However, the interference and/or the different amplification efficiencies among different primer sets often lead to biased amplification and thus limited multiplexing level. We here defined these methods as narrow-sensed multiplex LAMP. The research on miniaturized amplification technology which is booming in recent years has given rise to the novel general-sensed multiplex LAMP technology that breaks this limitation by its capability to perform highly parallel and miniaturized simplex reactions in independent compartments. Methods of this type have additional benefits such as lower reagent cost, higher level of automation, lower risk of cross-contamination and better suitability for on-site detection of multiple targets. In this review, we summarize the recent research progress in multiplex LAMP technology from the following aspects: the principle and design of narrow-sensed LAMP and its amplification optimization, the general-sensed LAMP, and the various applications of all multiplex LAMP technologies in diagnostics.

[Establishment of a hydrogel chip for high-throughput detection of Y chromosome microdeletions].

OBJECTIVE: To establish a high-sensitivity, high-specificity and low-cost hydrogel chip platform for the clinical screening of Y chromosome microdeletions. METHODS: Site-specific extended primers with a common sequence at the 5' end were used for hybridizing with the target. The Cy5-dUTP was incorporated into the products by primer extension, and the products were labeled with fluorescence. Then the extended products were added to the chip for hybridizing with acrylamide-modified common probes immobilized on the chip. After removal of the free Cy5-dUTP by electrophoresis, the signals were obtained by fluorescence scanning. And the detecting conditions of this method were optimized. RESULTS: SY254 of 9 samples was successfully detected with the hydrogel chip. The results showed that 3 were normal and the other 6 with microdeletions (1 female sample as a negative control), which coincided with the results of conventional multiplex PCR-electrophoresis. CONCLUSION: The hydrogel chip platform we established has provided a new technique for the detection of Y chromosome microdeletions, and is beneficial to the diagnosis and treatment of male infertility.

[Advances in approaches for the quantitative detection of microRNAs].

MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play an important role in the control of developmental process of different cells by negative regulation of protein-coding gene expression. Analyzing miRNA expression in tissues or cells can supply valuable information for investigating the biological function of these molecules. Recently, researchers had proposed a number of approaches for analyzing the differences of miRNA expression among different physiological or pathological conditions, and found that aberrant expression of miRNA was related to cancers, neurological disorders and heart diseases, etc. This review focuses on newly developed strategies for miRNA quantification, and elucidates in detail the probe-hybridization based methods including Northern blotting, microarray, gold nanoparticle labelling, and splinted ligation with radioactive labels. The amplification-based methods including quantitative PCR, rolling cycle amplification, invader assay, and the next generation sequencing methods were also discussed. The advantages and disadvantages of these methods were compared.