Near infrared (NTR)fluorescentacycloterniinators were tested as substrates in the Sanger enzymatic method ofDNA sequencing. The acyclic triphosphates ofadenosine, uridine, guanosine, and cytidine (AcyNTP) were labeled with a heptamethine carbocyanine dye via a propargylamino linker to the purine or pyrimidine base. Dye-labeled AcyNTPs which are lacking in the sugar moiety positions equivalent to the C-2 and C-3 ofthe ribose functioned similarly to chain-terminating dideoxynucleotides (ddNTPs). These fluorescent nonnucleotide analogs were incorporated by a mutant, thermostable Taq DNA polymerase with the same efficacy and fidelity as traditional ddNTPs. Sequence read length and basecalling accuracy were comparable for both dye-acyclotenninator and dye-primer sequencing methods. In two primer walking projects, cycle sequencing with fluorescent AcyNTPs achieved a mean sequence read length of 1,090 bases with 99. 1% accuracy at one kilobase read length. The cyanine dye-labeled acycloterminators produced electropherograms in which weak T peaks follow G peaks. In cases of polymorphism, such peak height variability may make it difficult to distinguish the presence or absence of a heterozygote at a specific site.

MagneSilTM paramagnetic particles allow the flexibility ofautomating the isolation ofDNA from as little as 20mg ofplant material to as much as 500 grains ofvegetable oil for use in testing for DNA sequences from genetically modified organisms (GMO), or plant breeding applications such as random amplification polymorphism detection (RAPD) or polymerase chain reaction (PCR). Given the wide variety of plant materials, foods and highly processed food ingredients that require testing, the purification system must be both scalable and flexible in its ability to purify DNA from such a wide array ofsample types. The procedures used in these purification systems are similar to other methods used for the walkaway automation ofplasmid purification and DNA sequencing reaction cleanup used in genomics applications, as well as DNA purification ofDNA from PCR reactions used for genetic interogations or DNA immobilizations. These purification systems can be used with a variety ofrobotic workstations in 96 well formats.

Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. We are addressing this challenge through instrumentation and approaches to rapidly express, purify, crystallize, and mutate large numbers of human gene products. Our approach applies the principles of HTS technologies commonly used in pharmaceutical development. Genes are cloned, expressed, and purified in parallel to achieve a throughput potential of hundreds per day. Our instrumentation allows us to produce tens of milligrams of protein from 96 separate clones simultaneously. Purified protein is used for several applications including a high-throughput crystallographic screening approach for structure determination using automated image analysis. To further understand protein function, we are integrating a mutagenesis and screening approach. By combining these key technologies, we hope to provide a fundamental basis for understanding gene function at the protein level.

Fourier transform mass spectrometry (FTMS) is increasingly being used as a drug discovery tool. We describe the development of a parallel high-throughput screening (HTS) strategy to identify small molecules that bind RNA targets using FTMS as an alternative to classical high-throughput biological screening methods for combinatorial libraries. The Multitarget Affinity/Specificity Screening (MASS) assay takes advantage of the "intrinsic mass" label of each compound and target RNA by employing high resolution, high precision mass measurements. The ability to analyze complex mixtures allows large compound libraries to be screened in the presence of multiple RNA targets simultaneously. The identity of the small molecule(s) which bind, the RNA target to which it binds, the compound-specific binding affinity and the location of the binding site on the RNA can be determined in one set of rapid experiments. The MASS technology detects complexes with dissociation constants of < 5 mM, with high sensitivity.

Facilities engaged in proteome analysis differ significantly in the degree that they implement automated systems for high-throughput protein characterization. Though automated workstation environments are becoming more routine in the biotechnology and pharmaceutical sectors of industry, university-based laboratories often perform these tasks manually, submitting protein spots excised from polyacrylamide gels to institutional core facilities for identification. For broad compatibility with imaging platforms, an optimized fluorescent dye developed for proteomics applications should be designed taking into account that laser scanners use visible light excitation and that charge-coupled device camera systems and gas discharge transilluminators rely upon UV excitation. The luminescent ruthenium metal complex, SYPRO Ruby protein gel stain, is compatible with a variety of excitation sources since it displays intense UV (280 nm) and visible (470 nm) absorption maxima. Localization is achieved by noncovalent, electrostatic and hydrophobic binding of dye to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream microchemical characterization techniques such as matrix-assisted laser desorption/ionization-mass spectrometry is assured. Protocols have been devised for optimizing fluorophore intensity. SYPRO Ruby dye outperforms alternatives such as silver staining in terms of quantitative capabilities, compatibility with mass spectrometry and ease of integration into automated work environments.

While the DNA chip is going to be prevalent for the medical diagnostics and drug development, its analysis tool such as a chip scanner need to be optimized. To obtain the guideline for the optimized CCD-based scanner, this paper addresses the minimum requirements for the selected CCD-based detector under the chosen light condition. The scope of our experiments was focused on the scanning performance rather than the design of the chip scanner itself. Through the gray level analysis of the target spot and background, we could check the possibility of the CCD-based detector and the lighting lamp to adopt them as a low price scanner. A quality measure was investigated with two levels of lighting power and the various integration times. For the CCD-based detector and the Xenon lighting system, we evaluated the image performance with the developed quality measure. We define a background and a sample area mask manually to investigate the statistics of the images captured by the scanner. The developed system is composed with a medium price CCD camera and a zenon lamp lighting system. The developed system has reasonable performance for the scanned image. The risk ofphotobleaching was not shown in our experiments.

The light-up probe is a recently developed probe for monitoring PCR amplification in real time. It is a peptide nucleic acid (PNA) coupled to an asymmetric cyanine dye that becomes fluorescent upon binding nucleic acids. The light-up probe is used to monitor product accumulation in regular three steps PCR. It is designed to bind target DNA at annealing temperature, where the fluorescent signal is recorded, and to dissociate at elongation temperature. Here we study the effect of experimental conditions on light-up probe monitored real-time PCR. In particular, we study the effects of Mg2, primer, dNTP, Taq and probe concentrations. We find that the light-up probe can be used to monitor product formation under a wide range of conditions. Lowest threshold cycle, reflecting minimal inhibition of the PCR reaction, and large fluorescence enhancement, reflecting efficient probe binding and substantial amount of product formation, was observed in 3 mM [Mg2] 10 mM, 0.4 tM [primer], 50 iM [dNTP] 600 jiM, 0.5 U [Taqi, using 0.2 iM [light-up probe] 1 riM. Some light-up probe fluorescence enhancement is observed in non-template controls (ntc), i.e., samples containing all PCR components but template, which gives rise to primer-dimer products. This signal has a distinct shape and it reaches lower amplitudes than signals from positive samples, which makes it readily distinguishable.

Molecular beacons (MBs) are novel nucleic acid hairpin-shaped probes that brightly fluoresce when they are bound to their targets and have a significant advantage over conventional nucleic acid probes. We have developed ultrasensitive MB DNA biosensors, including a fiber optic evanescent wave sensor and a submicrometer optical fiber sensor. The fiber optic sensors are rapid, stable, reproducible, regenerable and have the capability for one base mismatch discrimination. The sensors have been applied for both detection of DNA and specific mRNA sequences. A sensor array has also been demonstrated for multiple target identification. A microwell array methodology has been developed for ultrasmall amount DNA analysis, and can detect 50 DNA molecules in each well. Our study has also extended the applications of MBs from pure nucleic acid hybridization to DNA-protein interactions. These recent advancements of ultrasensitive DNA/protein analysis by molecular beacon probes provide a powerful tool for genomics and proteomics studies, molecular diagnoses of diseases, and new drug development.

HIV-1 viral load assays require accuracy and sensitivity at low RNA levels with the capability to detect all subtypes. Furthermore, the assay should be easy to perform and fast to be useful for routine diagnostics. In order to meet these demands we have combined isothermal NASBA amplification with molecular beacon probes for real-time detection and quantitation of HIV-1 RNA. Quantitation is based on co-amplification of the HIV-1 RNA in the clinical sample and a synthetic calibrator RNA which is amplified by the same primer set but detected with a differently labeled molecular beacon. The entire procedure is simple and analysis of 48 samples requires less than 1½ hours with minimal hands-on time. A fluorescent plate reader is used for real-time detection and isothermal amplification. The linearity and precision of the assay was determined with the VQC HIV-1 type B standard of the Central Laboratory of the Dutch Red Cross Blood Banks, The Netherlands. Sensitivity was shown to be 50 copies per ml (cps/ml). The average assay precision was 0,19 log10 over a range of 100-300,000 cps/ml tested at nine concentrations. The linearity of dilution series of 15 cultured HIV-1 gag clades A-H was shown. The specificity was 100% on non HIV-1 samples HIV-2, HTLV-1 and HTLV-2. The assay robustness in terms of valid results was 99%. In conclusion, the new real-time NASBA assay meets state-of-the-art HIV-1 viral load performance requirements combined with a high level of user convenience.

DNA cleavage reactions are important in both cellular processes and biotechnology. Conventional assays are discontinuous, time consuming and laborious, and there is a great demand for continuous, convenient and sensitive assays for these reactions. Here we introduce a novel method for the monitoring of the cleavage reactions in real time. Molecular beacon is used as a substrate for the single strand specific DNA nucleases. The cleavage of the molecular beacon by Si nuclease is demonstrated as an example for this method. The cleavage causes a great increase in fluorescence intensity of the molecular beacon, giving a signal-to-noise ratio of about 40. This assay is sensitive, continuous and easy to use. It can be applied to the detection of DNA nucleases, the characterization of these enzymes and for detailed study of the mechanisms of the cleavage reactions. The influence of solution conditions, such as macromolecular crowding, on the cleavage reactions has been studied.