Rapid DNA detection by beacon-assisted detection amplification

This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples.     

Alpha detection

The problem of defining EEG alpha activity, and of detecting and measuring it, is outlined. Some advantages of a threshold method are presented and compared with an amplitude-integration method. Characteristics of percent-time scoring, based upon observable alpha spindles in the waking EEG, are discussed. A probability distribution of percent-time alpha as a function of detection threshold is derived and compared with empirical data.     

Mycotoxin detection

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Fluorescence-based mutation detection

Conventional SSCP analysis of DNA amplified by polymerase chain reaction (PCR-SSCP) is one of the simplest and most reliable tools for identifying point mutations, and small insertions or deletions. The sensitivity of the technique is increased by using the Applied Biosystems (ABI) semiautomated DNA sequencer equipped with GENESCAN 672 software for F-SSCP. The four-dye ABI system permits a red dye-labeled internal lane standard to be run in the same lanes as the DNA being examined, leaving three dye colors for labeling DNA of interest. The internal lane standard is used to normalize gels or correct for minor differences in apparent electrophoretic mobility between lanes. Correction for these lane-dependent differences in migration and the capability to stack data from two different lanes on the computer screen makes it possible to detect sequence variants that produce very small mobility shifts. Coelectrophoresis of control and unknown DNA in the same lane, using different dye labels for each, is also helpful for detecting sequence variants that produce small mobility changes. Multiplexing multiple F-SSCP targets in the same lane increases sample throughput.     

Mutation detection in Machado-Joseph disease using repeat expansion detection.

BACKGROUND: Several neurological disorders have recently been explained through the discovery of expanded DNA repeat sequences. Among these is Machado-Joseph disease, one of the most common spinocerebellar ataxias (MJD/SCA3), caused by a CAG repeat expansion on chromosome 14. A useful way of detecting repeat sequence mutations is offered by the repeat expansion detection method (RED), in which a thermostable ligase is used to detect repeat expansions directly from genomic DNA. We have used RED to detect CAG expansions in families with either MJD/SCA3 or with previously uncharacterized spinocerebellar ataxia (SCA). MATERIALS AND METHODS: Five MJD/SCA3 families and one SCA family where linkage to SCA1-5 had been excluded were analyzed by RED and polymerase chain reaction (PCR). RESULTS: An expansion represented by RED products of 180-270 bp segregated with MJD/SCA3 (p < 0.00001) in five families (n = 60) and PCR products corresponding to 66-80 repeat copies were observed in all affected individuals. We also detected a 210-bp RED product segregating with disease (p < 0.01) in a non-SCA1-5 family (n = 16), suggesting involvement of a CAG expansion in the pathophysiology. PCR analysis subsequently revealed an elongated MJD/SCA3 allele in all affected family members. CONCLUSIONS: RED products detected in Machado-Joseph disease families correlated with elongated PCR products at the MJD/SCA3 locus. We demonstrate the added usefulness of RED in detecting repeat expansions in disorders where linkage is complicated by phenotyping problems in gradually developing adult-onset disorders, as in the non-SCA1-5 family examined. The RED method is informative without any knowledge of flanking sequences. This is particularly useful when studying diseases where the mutated gene is unknown. We conclude that RED is a reliable method for analyzing expanded repeat sequences in the genome.     

Better mutation detection

Mutation detection and typing of polymorphic loci through double-strand conformation analysisArgüello, J.R. et al. (1998)Nat. Genet. 18, 192–194     

Biomaterial-based infrared detection

This effort is focused on the use of crustacyanin protein extracted from the lobster shell in IR detection and imaging applications. In addition to the protein's excellent reversible thermo-active response in the IR region of interest, electrical characteristics versus temperature showed that the protein can be used as an electro-optic thermal sensing device as well. The high sensitivity and fast response of the protein layer were further enhanced by the deposition process we used. The thin coatings were prepared by Langmuir-Blodgett and self-assembly techniques. Furthermore, the protein exhibited temperature variation under Ti:sapphire laser excitation at different wavelengths in ambient environment. We have also shown that the protein exhibits fluorescence properties after exposure to IR heat. Stability of the protein, which is important in this type of application, was also demonstrated using the different characterization techniques after repeated heating/cooling cycles. We can conclude that this protein represents a formidable candidate for the fabrication of IR sensors and microbolometers for uncooled IR imaging applications.     

VisRD--visual recombination detection

SUMMARY: VisRD, a program for visual recombination detection in a sequence alignment is presented. VisRD is written in Java and is designed to complement the multi-purpose phylogenetic software package SplitsTree4. AVAILABILITY: The software is freely available from http://www.lcb.uu.se/~vmoulton/software/visrd/     

Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis

This minireview presents recent developments in molecular methods for the diagnosis of tuberculosis, including detection, identification and determination of drug resistance of Mycobacterium tuberculosis . Tuberculosis remains one of the major causes of global death from a single infectious agent. This situation is worsened by the HIV/AIDS pandemic because one-third of HIV/AIDS patients are coinfected with M. tuberculosis . Also of great concern is the emergence of drug-resistant tuberculosis because there are almost no treatment options available for patients affected by highly resistant strains of M. tuberculosis . Advances in molecular biology techniques and a better knowledge of the molecular mechanisms of drug resistance have provided new tools for the rapid diagnosis of tuberculosis. Several nucleic acid amplification technologies have been developed and evaluated. New molecular approaches are being introduced continuously. This minireview will also comment on the future perspectives for the molecular diagnosis of tuberculosis and the feasibility for the implementation of these newer techniques in the clinical diagnostic laboratory.     

Surface plasmon resonance-based detection of ladder-shaped polyethers by inhibition detection method

Ladder-shaped polyether (LSP) compounds represented by brevetoxins and ciguatoxins were largely discovered in association with seafood poisoning. Thus, a quick quantification method for LSPs is potentially important. We examined a surface plasmon resonance method using desulfated-yessotoxin (dsYTX) immobilized on a sensor chip and phosphodiesterase PDEII in a inhibition detection mode. Yessotoxin, brevetoxin B and synthetic LSP derivatives showed clear inhibition against PDEII binding to the immobilized dsYTX, by which their half inhibitory concentrations were successfully estimated. This inhibition method appeared to be superior in specificity to direct binding assays where binding proteins to LSP was immobilized on a sensor chip.     

Ligation detection reaction-TaqMan procedure for single nucleotide polymorphism detection on genomic DNA

In this article, we describe a genotyping approach applicable to both individual and multiplexed single nucleotide polymorphism (SNP) analysis, based on a ligation detection reaction (LDR) performed directly on genomic DNA. During the ligation, the biallelic state of the SNP locus is converted into a bimarker state of ligated detector oligonucleotides. The state of the markers is then determined by a 5'-nuclease assay (TaqMan) with universal fluorescent probes. The LDR-TaqMan method was successfully applied for the genotyping of 30 SNP loci of Arabidopsis thaliana. The technology is cost-effective, needs no locus-specific optimization, requires minimal manipulations, and has very good potential for automation.     

Tendon extracellular matrix damage detection and quantification using automated edge detection analysis

The accumulation of sub-rupture tendon fatigue damage in the extracellular matrix, particularly of type I collagen fibrils, is thought to contribute to the development of tendinopathy, a chronic and degenerative pathology of tendons. Quantitative assessment of collagen fibril alignment is paramount to understanding the importance of matrix injury to cellular function and remodeling capabilities. This study presents a novel application of edge detection analysis to calculate local collagen fibril orientation in tendon. This technique incorporates damage segmentation and stratification by severity which will allow future analysis of the direct effect of matrix damage severity on the cellular and molecular response.