Pullulan reduces the non-specific amplification of loop-mediated isothermal amplification (LAMP)

Loop-mediated isothermal amplification (LAMP) has been developed as a versatile method for nucleic acid analysis in many applications. However, non-specific LAMP leading to false-positive outcomes has been observed frequently. To solve this problem, we selected six molecules as the additives for evaluating their effects on the improvement of the LAMP specificity. Experimental results show that bovine serum albumin (BSA) and DL-dithiothreitol (DTT) have negative effects on the LAMP specificity; dimethyl sulfoxide (DMSO), tetramethylene sulfoxide (TMSO), and glycerol could inhibit the non-specific LAMP moderately. Surprisingly, pullulan shows an ability to inhibit the non-specific amplification of LAMP significantly without affecting the sample amplification of LAMP, and this inhibitory effect is concentration dependent. Thus, pullulan could be considered as the most promising additive to improve the amplification specificity in the LAMP-based detection and analysis of nucleic acids.

     

Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.     

Sensing through signal amplification

The naked eye detection of single molecules in a complex mixture is the ultimate detection limit. Since a single molecule is unable to generate a strong enough signal, sensing methodologies able to reach that limit by necessity need to rely on signal amplification. This tutorial review describes various molecular approaches towards signal amplification in which a single analyte molecule affects the properties of a multitude of reporter molecules. Sensing by advanced instrumentation or changes in the physical properties of materials are excluded. The review is divided into four parts (catalysts, macromolecules, metal surfaces and supramolecular aggregates) depending on the species responsible for generating reporter molecules. Although on first sight apparently very diverse in nature, the majority of approaches rely on two key concepts: catalysis and multivalency. The ability of a catalyst to convert a multitude of substrate molecules into product (defined by the turn over number) makes a catalyst an intrinsic signal amplifier in case the chemical conversion of the substrate is accompanied by a measurable change in physical properties. For sensing purposes, catalytic activity must depend on the interaction between the analyte and the catalyst. Sensing using multivalent structures such as polymers and functionalized nanoparticles relies on the ability of a single analyte molecule to affect the properties of a multitude of reporter molecules collected in the multivalent structure. Chemical sensing systems will be discussed with detection limits that indeed go down to a few molecules and can rival the best biological assays. It will be shown that the most sensitive methods rely on a cascade of amplification mechanisms.     

Fluorometric determination of microRNA based on strand displacement amplification and rolling circle amplification

The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.

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Graphical abstract Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.

     

Signal amplification by rolling circle amplification on universal flaps yielded from target-specific invasive reaction

Detection of nucleic acids with signal amplification is preferable in clinical diagnosis. A novel approach was developed for signal amplification by coupling invasive reaction with hyperbranched rolling circle amplification (HRCA). Invasive reaction, which does not rely on specific recognition sequences in a target but a specific structure formed by the specific binding of an upstream probe and a downstream probe to a target DNA, can generate thousands of flaps from one target DNA; then the flaps are ligated with padlock probes to form circles, which are the templates of HRCA. As HRCA amplicon sequence is free of target DNA sequence, signal amplification is achieved. Because flap sequence is the same to any target of interest, HRCA is universal; the detection cost is hence greatly reduced. The sensitivity of the proposed method is less than 1 fM artificial DNA targets; and the specificity of the method is high enough to discriminate one base difference in the target sequence. The feasibility was verified by detecting real biological samples from HBV carriers, indicating that the method is highly sensitive, cost-effective, and has a low risk of cross-contamination from amplicons. These properties should give great potential in clinical diagnosis.     

High amplification cobalt imaging system

A novel high gain photothermographic imaging process is described. The essential components consist of a Co^III amine, a redox transfer lig- and L and a light-sensitive element capable of producing centers of Co^II. On exposure, a reducing agent is produced, commonly via a quinone which photogenerates a hydroquinone. On heating, the hydroquinone reduces Co^III amine to produce Co^II. The ligand then complexes this Co^II to form Co^II L_n, which, in turn reduces more Co^III amine. The resulting reaction produces a metallized dye, Co^IIIL_n, amine and more Co^II, which in the presence of excess L continues the cycle, giving photographically useful amplification. The system is capable of speed several thousand times that of an efficient one-quantum process.     

Signal amplification by allosteric catalysis

In this article we unify a series of recent studies on bio- and chemosensors under a single signaling strategy: signal amplification by allosteric catalysis (SAAC). The SAAC strategy mimics biological signal transduction processes, where molecular recognition between an external signal and a protein receptor is allosterically transduced into catalytically amplified chemical information (usually second messengers). Several recent biosensing and chemosensing studies apply this nature-inspired strategy by using engineered allosteric enzymes, ribozymes, or regulatable organic catalysts. The factors pertinent to achieving high sensitivity and specificity in SAAC strategies are analyzed. The authors believe that these early studies from a variety of research groups have opened up a new venue for the development of sensing technologies where molecular recognition and catalysis can be coupled for practical purposes.     

Hydrophobic amplification of noncovalent organocatalysis

The effects of hydrogen-bonding organocatalysts and water for the acceleration of epoxide openings with a variety of nucleophiles are additive and lead to excellent yields of the catalyzed reactions in water.     

Massively parallel display of genomic DNA fragments by rolling-circle amplification and strand displacement amplification on chip

Massively parallel genomic DNA fragments display on chip plays a key role in the new generation DNA sequencing. Here, we developed a new technology to display the parallel genomic DNA fragment massively based on two-step reaction with Ф29 DNA polymerase. The genomic DNA fragments were firstly amplified by rolling-circle amplification (RCA) reaction in liquid phase, and then amplified further on the chip by the strand displacement of Ф29 DNA polymerase. In our experiments, through DNA colonies produced by two-step amplification reaction T7 genomic DNA fragments are displayed massively and parallely on the chip, which has been verified through hybridizing the probe labeled with fluorescence or extension reaction with fluorescent-dNTP. The significant difference of fluourescence signals between background and displayed DNA fragments could be obtained. Our results show that the method has good reproducibility in experiments, which may be hopeful to serve the high-throughput sequencing.     

Serine sublimes with spontaneous chiral amplification

Sublimation of near-racemic samples of serine yields a sublimate which is highly enriched in the major enantiomer; this simple one-step process occurs under relatively mild conditions, and represents a possible mechanism for the chiral amplification step in homochirogenesis.     

Surface amplification of invasive cleavage products

A major focus of current efforts in genomics is to elucidate the genetic variations extent within the human population, and to study the effects of these variations upon the human system. The most common type of genetic variations are the single nucleotide polymorphisms (SNPs), which occur every 500-1000 nt in the genome. Large-scale population association studies to study the biological or medical significance of such variations may require the analysis of hundreds of thousands of SNPs on thousands of individuals. We are pursuing development of an approach to large-scale SNP analysis that combines the specificity of invasive cleavage reactions with the parallelism of high density DNA arrays. A surface-immobilized probe oligonucleotide is specifically cleaved in the presence of a complementary target sequence in unamplified human genomic DNA, yielding a 5' phosphate group. High sensitivity detection of this reaction product on the surface is achieved by the use of rolling circle amplification, with an approximate concentration detection limit of 10 fM target DNA. This combination of very specific surface cleavage and highly sensitive surface detection will make possible the rapid and parallel analysis of genetic variations across large populations.     

DNAzyme amplification of molecular beacon signal

This paper reports an improved catalytic molecular beacon. Addition of the target oligonucleotide activates a DNA enzyme (DNAzyme), which, in turn, activates multiple copies of molecular beacons (MB) and gives rise to a strong fluorescence signal. In a previous design, the activated DNAzyme could oligomerize, especially dimerize, and result in inactivation of the DNAzyme. The current design avoids this problem, upon activated by the target DNA, the DNAzyme will stay constantly active. With the improved method, a detection of 10 pM DNA has been demonstrated, which is 1000 times more sensitive than the method previously reported.     

Cascade signal amplification strategy for the detection of cancer cells by rolling circle amplification and nanoparticles tagging

A cascade signal amplification strategy was proposed for detection of cancer cells at ultralow concentration by combining the rolling circle amplification (RCA) technique with oligonucleotide functionalized nanoparticles (NPs), and anodic stripping voltammetric detection. This flexible biosensing system exhibited high sensitivity and specificity with the detection limits of 10 Ramos cells mL(-1).     

Ultrasensitive electrochemical immunoassay based on counting single magnetic nanobead by a combination of nanobead amplification and enzyme amplification

An ultrasensitive electrochemical immunoassay method based on counting single magnetic nanobead (MNB) with combined amplification of nanobead and enzyme. Carcinoembryonic antigen (CEA) and MNB were initially immobilized on substrate at 1:1 molar ratio through sandwich immunoreactions. The MNBs were then labeled with alkaline phosphatase (AP) and continuously introduced to capillary with disodium phenyl phosphate (DPP). APs convert DPPs into a phenol zone around each moving MNB, i.e., one CEA. The phenol zones can be electrochemically detected as peaks and counted for CEA quantification. The detection limit for CEA is 5.0 × 10^?17 mol/L.     

Raman amplification spectroscopy using pulsed dye lasers

A picosecond laser system consisting of a mode-locked argon-ion laser synchronously pumping two dye lasers is used for studies of Raman amplification spectra. The two dye laser beams, one kept constant in frequency while the other is tunable, coincide in the Raman sample. Recording the gain or the loss in intensity of one of the lasers as a function of frequency difference produces the Raman spectrum. Good signal to noise ratios have been obtained for a variety of liquids and solids. Fluorescing samples can be studied in the Inverse Raman method where the loss on the higher frequency laser is monitored.     

Iodometric microdetermination of semicarbazide by amplification reactions

An analytical method for the determination of 10–2000 μg of semicarbazide is described, using titrimetric procedure with an amplification reaction. It relies upon the reaction of semicarbazide, in phosphate-buffer (pH 7) solution, with a chloroform solution of iodine, and removal of its excess; oxidation of the iodide formed with bromine; and determination of the liberated iodate by the Leipert amplification procedure. The recovery ranged from 97.0 to 100.1%.     

Signal amplification and transduction by photo-activated catalysis

A simple flavin-based catalytic system is able to transform light into chemical output with amplified response utilizing a Cu(I)-catalyzed cycloaddition reaction.