DNA probes

Simple, low-cost and accurate genotyping methods for single nucleotide polymorphisms (SNPs) are in high demand in the post-genome-sequencing era. We present a graphical tool called SNPicker, implemented in Java, which significantly facilitates the design of mutagenic endonuclease restriction assays. SNPicker uses the online NEB REBASE to automatically scan for all possible designs of mutagenic primers that can facilitate the picking of mismatched PCR primers to artificially introduce or abolish a restriction site at the target SNP site. We successfully applied SNPicker in designing endonuclease restriction assays for 14 SNPs for the MTHFR gene, the Coagulation Factor II gene and the Coagulation Factor V gene. The SNP assays designed using SNPicker were cross-validated using the MassARRAY technology.

Efficient DNA sequencing of the genomes of individual species and organisms is a critical task for the advancement of biological sciences, medicine and agriculture.Advances in modern sequencing methods are needed to meet the challenge of sequencing such megabase to gigabase quantities of DNA. Two possible strategies for DNA sequencing exist: direct methods, in which each base position in the DNA chain is determined individually (e.g., gel sequencing or pyrosequencing), and indirect methods, in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the DNA chain. One promising indirect method is sequencing by hybridization (SBH), in which sets of oligonucleotides are hybridized under conditions that allow detection of complementary sequences in the target nucleic acid. The unprecedented sequence search parallelism of the SBH method has allowed development of high-throughput, low-cost, miniaturized sequencing processes on arrays of DNA samples or probes. Newly developed SBH methods use DNA ligation to combine relatively small sets of short probes to score potentially tens of millions of longer oligonucleotide sequences in a target DNA. Such combinatorial approaches allow analysis of DNA samples of up to several kilobases (several times longer than allowed by current direct methods) for a variety of DNA sequence analysis applications, including de novo sequencing, resequencing, mutation/SNP discovery and genotyping, and expression monitoring. Future advances in biochemistry and implementation of detection methods that allow single-molecule sensitivity may provide the necessary miniaturization, specificity, and multiplexing efficiency to allow routine whole genome analysis in a single solution-based hybridization experiment.

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to labelfree and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples.

Most studies on preconception diagnosis published so far have used polymerase chain reaction (PCR) analysis to identify single gene defects. Although fluorescent DNA probes have been used to obtain a partial cytogenetic diagnosis of aneuploidies in first polar bodies without defined chromosome structures, the analysis of structural chromosome anomalies in the interphase nucleus is not adequate. We describe a procedure to obtain first polar body chromosome complements from hamster and human oocytes. In 63.6% (105 of 165) of hamster first polar bodies the chromosome complement showed a defined chromosome morphology and in 94.1% (16 of 17) of human oocytes fixed after follicular puncture it was possible to obtain high quality, well spread chromosome complements. First polar body chromosomes are fuzzy and shorter than oocyte chromosomes, but fluorescent in-situ hybridization results obtained in human first polar bodies clearly show that it is possible to detect whole chromosomes, centromeres and unique sequences, including the terminal regions of small chromosomes. This suggests that in fresh oocytes, DNA loss resulting from apoptotic chromosome fragmentation has not yet occurred. Using the procedure described, first polar bodies could be used to analyse the meiotic segregation of maternal structural abnormalities and to detect numerical chromosome anomalies in humans.

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6)-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6)-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.

Array-based genome-wide screening methods were recently introduced to clinical practice in order to detect small genomic imbalances that may cause severe genetic disorders. The continuous advancement of such methods plays an extremely important role in diagnostic genetics and medical genomics. We have modified and adapted the original multiplex amplifiable probe hybridization (MAPH) to a novel microarray format providing an important new diagnostic tool for detection of small size copy-number changes in any locus of human genome. Here, we describe the new array-MAPH diagnostic method and show proof of concept through fabrication, interrogation and validation of a human chromosome X-specific array. We have developed new bioinformatic tools and methodology for designing and producing amplifiable hybridization probes (200 -600 bp) for array-MAPH. We designed 558 chromosome X-specific probes with median spacing 238 kb and 107 autosomal probes, which were spotted onto microarrays. DNA samples from normal individuals and patients with known and unknown chromosome X aberrations were analyzed for validation. Array-MAPH detected exactly the same deletions and duplications in blind studies, as well as other unknown small size deletions showing its accuracy and sensitivity. All results were confirmed by fluorescence in situ hybridization and probe-specific PCR. Array-MAPH is a new microarray-based diagnostic tool for the detection of small-scale copy-number changes in complex genomes, which may be useful for genotype -phenotype correlations, identification of new genes, studying genetic variation and provision of genetic services.

The interaction between DNA and several newly synthesized derivatives of the natural anticancer compound luotonin A has been studied. The results from our work reveal an effective and selective alkaloid/double-stranded DNA (ds-DNA) interaction. In the presence of increasing amounts of ds-DNA, a noticeable fluorescence quenching of the luotonin A derivatives under study was observed. However, this effect did not take place when singlestranded DNA (ss-DNA) was employed. The association constant alkaloids/ds-DNA was calculated by quantitation of such a quenching effect. The influence of other quenchers, namely Co 2+ and Br − on the native fluorescence of luotonin A and derivatives was also studied, and a remarkable quenching effect was observed for both ions. We have also investigated how by binding DNA the alkaloids could get protected from the external Co 2+ and Br − quenchers. The Stern-Volmer constants (K SV ) for Co 2+ and Br − quenching effect on the studied alkaloids were considerably reduced (10-50%) after incubation of the compounds in the presence of DNA with regard to the K SV values in absence of DNA. An increase in the fluorescence anisotropy values of luotonins was also produced only in the presence of ds-DNA but not in the case of ss-DNA. To better characterize the nature of that interaction, viscosimetry assays and ethidium bromide displacement studies were conducted. With regard to DNA reference solutions, the viscosity of solutions containing DNA and luotonin A derivatives was reduced or not significantly increased. It was also observed that the studied compounds were unable to displace the intercalating agent ethidium bromide. All of these results, together with the obtained association constants values (K ass =2.2×10 2 -1.3× 10 3 ), support that neither covalent nor intercalating interactions luotonin A derivatives/ds-DNA are produced, leading to the conclusion that these alkaloids bind ds-DNA through the minor groove. The specific changes in the fluorescence behavior of luotonin A and derivatives distinguishing between ss-DNA and ds-DNA binding, lead us to propose these compounds as attractive turn-off probes to detect DNA hybridization.

Figure 10. UV-vis spectra (A) and TEM images and size distributions (B) of (a) [AuCl 4 ]before reduction; dodecanethiol-AuNPs (b) as prepared and after heat treatment at (c) 150, (d) 190, and (e) 230°C; and (f) octadecanethiol-AuNPs heat-treated at 250°C. Reprinted with permission from ref 188b (Miyake's group).

The promoter region of the murine branched-chain α-ketoacid dehydrogenase E2 subunit (dihydrolipoyl transacylase) gene was isolated and characterized. Sequence analysis of the promoter-regulatory region showed the presence of two inverted ‘CAAT box’ sequences, the most proximal being −42 to −48 bp upstream from the determined transcription initiation site, but no TATA-box sequences, similar to the human BCKAD E2 gene. The boundary of the minimum promoter sequence appeared to reside just inclusive of this first inverted CAAT sequence, but minigene transfer analysis demonstrated that the promoter proximal region between −65 and −140 bp is likely to be extremely important for controlling regulated changes in E2 RNA expression. The regulatory effect of this region may be modulated by a number of other upstream regions which were identified within the 7.0 kb sequence examined.

Background: With multiple strains of various pathogens being sequenced, it is necessary to develop high-throughput methods that can simultaneously process multiple bacterial or viral genomes to find common fingerprints as well as fingerprints that are unique to each individual genome. We present algorithmic enhancements to an existing single-genome pipeline that allows for efficient design of microarray probes common to groups of target genomes. The enhanced pipeline takes advantage of the similarities in the input genomes to narrow the search to short, nonredundant regions of the target genomes and, thereby, significantly reduces the computation time. The pipeline also computes a three-state hybridization matrix, which gives the expected hybridization of each probe with each target.

Regulated antisense RNA enables the construction of a defined set of conditional growthdefective/lethal strains. In this study, we expanded the regulated antisense RNA interference technology and developed a high-throughput screening strategy to identify the potential drug targets of novel antimicrobials. To prove this concept, the specific antisense sublibrary of different essential open reading frames were pooled in the presence of an inducer, and treated with or without sublethal levels of mupirocin, triclosan, or gentamicin. Antisense RNA-expressing strains that were sensitized for increased susceptibility to the antibiotics were selectively detected via DNA subtractive hybridization, microarray, and whole-cell analyses. No strain was identified as supersensitive to gentamicin because there was no target-specific antisense strain in this sublibrary. In contrast, strains expressing antisense to isoleucine tRNA synthetase (ileS) and enoyl-[acyl-carrier-protein] reductase (fabI) were specifically identified as having increased susceptibility to mupirocin and triclosan, respectively. These results demonstrated that ileS and fabI antisense strains showed significant increases of susceptibility only to their specific inhibitors. This data demonstrates that a regulated antisense RNA expression library provides an effective tool to assist in the identification of potential targets for novel antibacterial agents. Highlights

Potentiometric stripping analysis is employed for highly sensitive measurements of single-stranded DNA at carbon paste electrodes. Electrochemical (oxidative) pretreatment is used to condition the carbon paste surface prior to the accumulation process. The low background response of the potentiometric stripping mode offers convenient quantitation of low nanogram quantities of DNA. Experimental variables of the pretreatment and measurement procedures are explored and optimized. Results are also reported for the quantitation of nanogram levels of the long terminal repeat (LTR) from the human leukemia virus and for the stripping operation of microfabricated screen-printed electrodes.

The synthesis of ribosomes is a major metabolic activity critical for cell growth and homeostasis. Understanding the mechanisms of ribosome biogenesis has important implications for studying both protein synthesis and cell cycle control. This unit describes several techniques for the analysis of rRNA maturation and ribosome assembly adapted for mammalian cells. Metabolic labeling of rRNA and hybridization analysis of precursors can be used to assess changes in rRNA processing that occur under experimental conditions of interest. Separation of preribosomal particles by sucrose gradient centrifugation is suitable for the analysis of proteins associated with preribosomes during their assembly and maturation in the cell nucleus.

The genetic relationships between several wild species and subspecies of the genus Hordeum were assessed using fluorescence in situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species, H. marinum ssp. maHnum (2n=14) and gussoneanum (2n = 14), and H. murinum ssp. murinum (2n = 28), and leporinum (2n = 28) and the" perennial species H. bulbosum (2n=14) and H. secalinum (2n=28), plus the South American perennial species H. chilense (2n-14). FISH was used to locate the chromosomal sites of two rDNA multigene families 5S and 18S-26S (pTa71 and pTa794) and three repetitive DNA sequences (pSc119.2, pAsl and pHch950) isolated from different species and genera. The seven chromosomes of the diploid species were readily distinguished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAsl. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa~ The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin of H. murinum and H. secelinum. The in situ experiments yielded the following conclusions: (1) differences between the subspecies marinum and gussoneanum; (2) close relationships between the subspecies murinum and leporinum; and (3) major differences in physical mapping between H. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.

Fluorescence in situ hybridization (FISH) is becoming popular in the diagnosis of clonal chromosomal abnormalities. We set up a fast FISH procedure using an extensive set of specific probes. Conventional banding analysis (CBA) and FISH were compared in 260 newly diagnosed acute myeloid leukemia (AML) patients. For FISH the following probes were used: MLL, CBF-b/MYH11, ETV-6/AML1; AML1/ETO, BCR/ABL, PML/RAR, c-MYC, TP53, RB1, 5q31/5p15.2, 5q33-34, 7q31/CEP7 , 20q13; CEP 4, X, Y. Result time was 96 h for CBA versus 5 h for FISH from direct harvest. CBA showed clonal abnormalities in 41% (n ¼ 105/ 260), normal karyotype in 39% (n ¼ 102/260) and failed in 20% (n ¼ 53/260). FISH screened all patients and detected abnormalities in 39% (n ¼ 102/260); CBA and FISH together identified abnormalities in 49% (n ¼ 128/260). In six patients with normal CBA and in eight patients with clonal karyotype, it detected further cryptic abnormalities. CBA showed clonal abnormalities in 13% of patients negative at FISH (n ¼ 21/158). FISH screening does not add relevant information to CBA, but is the quickest method for detecting major genetic abnormalities in AML. The speed of FISH is very valuable in AML-M3/M3v because PML/RAR þ patients require specific therapy. Furthermore, we suggest FISH screening in failed, complex or suboptimal quality chromosome and specific FISH analysis for 5q, 7q, 12p, 17p, inv(16), t(11q23) in order to implement CBA accuracy.

The genome of the zebrafish, Danio rerio, contains two major classes of tandem repetitive elements (AT-rich and GC-rich). The AT-rich repeats can be further subdivided into two subgroups which differ by about 10% of 185 bp in the repeating unit. The chromosomal location of these sequences and the moderately repetitive 5S rDNA sequences was determined in two diploid zebrafish cell lines using in-situ hybridization with fluorochrome-labeled probes. The AT-rich sequences were found at the centromeres of all chromosome pairs and the GC-rich sequences were found in paracentromeric location on over half of the chromosomal pairs. Different patterns of hybridization were found for the two subgroups of the AT-rich family. One type hybridized primarily to centromeres of one half to two thirds of the chromosomal pairs and the other type to centromeres of about three fourths of the chromosomal pairs. The pattern of hybridization with the GC-rich sequences varied somewhat between the cell lines ...

Background: In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results: We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP-oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP-oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNPoligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without any cross reaction with non-Escherichia coli strains. Conclusion: This work gives entry into a new class of DNA/gold nanoparticles hybrid materials which might have optical property that can be controlled for application in diagnostics. We note that it should be possible to extend this strategy easily for developing new types of DNA biosensor for point of care detection. The salient feature of this approach includes low-cost, robust reagents and simple colorimetric detection of pathogen.

In Taiwan, 3 viral isolates were inade from a disease outbreak in rainbow trout Oncorhynchus rnykiss cultured in ponds during the penod from November 1994 to February 1995 Polymerase chain reaction (PCR) was used for identification of the isolates The results revealed that a specific 252 bp viral nucleotide was amplified by using infectious hematopoletic neci-osis virus (IHNV) specific primers. At the same time, several IHNV monoclonal antibodies (MAbs), and an IHNV specific rabbit polyclonal antibody were used to confirm the results of the PCR assay The results of the above assays indicated that all isolates were IHNV.

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

Infection of poultry with highly pathogenic avian influenza virus (AIV) can be devastating in terms of flock morbidity and mortality, economic loss, and social disruption. The causative agent is confined to certain isolates of influenza A virus subtypes H5 and H7. Due to the potential of direct transfer of avian influenza to humans, continued research into rapid diagnostic tests for influenza is therefore necessary. A nucleic acid sequence-based amplification (NASBA) method was developed to detect a portion of the haemagglutinin gene of avian influenza A virus subtypes H5 and H7 irrespective of lineage. A further NASBA assay, based on the matrix gene, was able to detect examples of all known subtypes (H1-H15) of avian influenza virus. The entire nucleic acid isolation, amplification, and detection procedure was completed within 6 h. The dynamic range of the three AIV assays was five to seven orders of magnitude. The assays were sensitive and highly specific, with no cross-reactivity to phylogenetically or clinically relevant viruses. The results of the three AIV NASBA assays correlated with those obtained by viral culture in embryonated fowl's eggs.

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets fromHaemophilus ducreyi,Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR

Nucleic acid probe-based assays are now widely used in genetic research, human identification, forensics and in a broad spectrum of clinical assays in the fields of microbiology, haematology/ oncology and virology. Labelled probes are used in a variety of assay formats including dot-blots, Southern blots (DNA target), Northern blots (RNA target), Western blots (protein target), in situ hybridization, plaque or colony screening and immobilized arrays on silicon or glass surfaces. Traditionally, the probes used in these assays have a radioactive 32phosphorous label that has a short shelf-life, is dangerous, has high disposal costs and, when labelled to high specific-activity, may be unstable. Extensive efforts to develop alternative labelling techniques have resulted in colorimetric, chemiluminescent and fluorescent assay formats. This review summarizes the properties desired in a probe, describes the advantages and disadvantages of the different nonradioactive labelling strategies, and illustrates examples of probe-based assays in which detection is facilitated by imaging samples using a general purpose fluorescence scanner.

Figure 10. UV-vis spectra (A) and TEM images and size distributions (B) of (a) [AuCl 4 ]before reduction; dodecanethiol-AuNPs (b) as prepared and after heat treatment at (c) 150, (d) 190, and (e) 230°C; and (f) octadecanethiol-AuNPs heat-treated at 250°C. Reprinted with permission from ref 188b (Miyake's group).

A mild and efficient surface activation protocol to convert polycarbonate (PC) substrates, e.g., plastic bases of compact disks, to biochip platforms for DNA probe immobilization and target detection is described. The preparation procedure (activation, patterning, and coupling) is simple and effective; the on-chip hybridization is sensitive and selective. Particularly, UV/ozone treatment of PC sheets produces a hydrophilic surface with a high density of reactive carboxylic acid groups [(4.8 ( 0.2) × 10 -10 mol/cm 2 ] in less than 10 min at ambient conditions, and no significant aging or physical damage to the substrate is observed. Covalent immobilization of DNA probes via both passive (reagent-less photopatterning and coupling in bulk solution phase) and flow-through (creation of microarrays with microfluidic channel plates) procedures has been demonstrated. Subsequent hybridization shows uniform and strong fluorescent signals for complementary target DNA and allows clear discrimination between fully complementary targets and strands with a single base-pair mismatch. The surface chemistry described herein will facilitate the development of disposable plastic biochips (not limited to DNA microarrays) and the fabrication of biomedical devices that are readable with conventional optical drives.

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to accurately enumerate a minimum of 3000 viable E. coli among a large number of non-fecal bacteria. When applied to river water and wastewater samples, the DVC-FISH method gave systematically higher E. coli counts than a reference culturebased method (miniaturized MPN method). The ratio between both counts (DVC-FISH/MPN) increased with decreasing abundance of culturable E. coli indicating that the proportion of viable but non-culturable (VBNC) E. coli (detectable by the DVC-FISH procedure and not by a culture-based method) was higher in low contaminated environments. We hypothesized that the more stressing conditions, i.e. nutritional stress and sunlight effect, met in low contaminated environments were responsible for the larger fraction of VBNC E. coli. A survival experiment, in which sterile mineral water was inoculated with a pure E. coli strain and incubated, confirmed that stressing conditions induced the apparition of non-culturable E. coli detectable by the DVC-FISH procedure. The analysis of the E. coli concentration along a Seine river longitudinal profile downstream a large input of fecal bacteria by a WWTP outfall showed an increasing fraction of VBNC E. coli with increasing residence time of the E. coli in the river after release. These data suggest that the DVC-FISH method is useful tool to analyze the dynamics of fecal bacteria in river water. D

Viral infection poses a major problem for public health, horticulture, and animal husbandry, possibly causing severe health crises and economic losses. Viral infections can be identified by the specific detection of viral sequences in many ways. The microarray approach not only tolerates sequence variations of newly evolved virus strains, but can also simultaneously diagnose many viral sequences. Many chips have so far been designed for clinical use. Most are designed for special purposes, such as typing enterovirus infection, and compare fewer than 30 different viral sequences. None considers primer design, increasing the likelihood of cross hybridization to similar sequences from other viruses. To prevent this possibility, this work establishes a platform and database that provides users with specific probes of all known viral genome sequences to facilitate the design of diagnostic chips. This work develops a system for designing probes online. A user can select any number of different viruses and set the experimental conditions such as melting temperature and length of probe. The system then returns the optimal sequences from the database. We have also developed a heuristic algorithm to calculate the probe correctness and show the correctness of the algorithm. (The system that supports probe design for identifying viruses has been published on our web page http://bioinfo.csie.ncu.edu.tw/.) Index Terms-Database, probe design, virus identification. I. INTRODUCTION V IRAL infection poses a major problem for public health, horticulture, and animal husbandry, possibly causing severe health crises and economic losses. Viral infections can be identified by the specific detection of viral sequences in two ways: the first is an amplification-based method, such as using

The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1-9) × 10 12 /cm 2 . The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 × 10 12 /cm 2 , electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.

Neuronal nicotinic receptors (nAchRs) have been isolated or cloned in insect, bird and mammalian neurons, but no information exists on the primary structure of human neuronal nAchRs. By screening a cDNA library from the human neuroblastoma cell line IMR 32 with a cDNA probe corresponding to the full length of rat ~t3-nicotinic subunit, we have identified an open reading frame encoding a protein of 502 amino acids. This protein shows all the features of members of the ligand-gated receptor superfamily and has two cysteine residues at positions 192, 193 which are typical of the nicotinic ~-subunits. Because of its high homology to rat ct3 (93% amino acid identity), we conclude that we have cloned the human ct3-nicotinic subunit.

Figure 10. UV-vis spectra (A) and TEM images and size distributions (B) of (a) [AuCl 4 ]before reduction; dodecanethiol-AuNPs (b) as prepared and after heat treatment at (c) 150, (d) 190, and (e) 230°C; and (f) octadecanethiol-AuNPs heat-treated at 250°C. Reprinted with permission from ref 188b (Miyake's group).

The early history of 'DNA fingerprinting' in the UK might have been different were it not for the accounts of two dramatic courtroom trials, made by the participants and the media, in the mid-1980s. But these reports, which misrepresented the importance DNA evidence had in the trials, left a strong impression on the British public and on judges on both sides of the Atlantic. These trials, widely considered to be the first 'victories' for DNA fingerprinting, have been frequently cited as proof of the utility and reliability of the technique, in both the UK and beyond. But in reality, it was the threat of DNA evidence being used rather than the integrity or validity of it that resolved these cases. At that time, DNA fingerprinting was still in its infancy, an untried and untested technology.

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

A Real-Time PCR (RT-PCR) assay was developed using TaqMan minor groove binder (MGB) probes for the specific detection and quantification of five acetic acid bacteria (AAB) species (Acetobacter pasteurianus, Acetobacter aceti, Gluconacetobacter hansenii, Gluconacetobacter europaeus and Gluconobacter oxydans) in wine and vinegar. The primers and probes, designed from the 16S rRNA gene, showed good specificity with the target AAB species. The technique was tested on AAB grown in glucose medium (GY) and inoculated samples of red wine and wine vinegar. Standard curves were constructed with the five target species in all these matrices. Quantification was linear over at least 5 log units using both serial dilution of purified DNA and cells. When this technique was tested in GY medium and inoculated matrices, at least 10 2 -10 3 cells/ml were detected. To quantify low populations of AAB in microbiologically complex samples, a PCR enrichment including part of the 16S-23S rRNA gene ITS region was needed to increase the amount of target DNA compared to non-target DNA.

RNAs are present within eukaryotic cells and are involved in several biological processes. RNA transport within cell compartments is important for proper cell function. To understand in depth the cellular processes in which RNA is involved requires a method that reveals RNA localization in real time in a sub-cellular context in living cells. In this protocol we describe a method for imaging RNA in living cells and in particular in neuronal cultures based on cell microinjection of molecular beacons in conjunction with confocal microscopy. This methodology overcomes some of the main obstacles for imaging RNA in live cells since microinjection allows the delivery of the probe to a desired cellular compartment and MBs bind with high specificity to its target RNA without inhibiting its function. The proper design of the MBs is essential to obtain RNA-MB association at the temperature of the cell cytosol. MBs design with other purposes in mind (such as PCR experiments) have a design that facilitates association to its target at high temperatures, rendering them unsuitable for live cell imaging. Using the methodology described in this chapter allows the study of RNA transport to different regions of neurons and may be combined with the tagging of proteins of interest to measure co-transport of the protein and the RNA to different cellular regions.

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 10 4 cells. Fusobacterium nucleatum ss polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.