Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Wavelength shifting oligonucleotide probe for the detection of adenosine of a target DNA with enhanced fluorescence signal

The modulated photophysical property of strong electronically coupled naphthyl uridine linked via a single C–C bond was explored in DNA detection via wavelength shifting and enhanced fluorescence emission by a simple ‘Just-Mix & Read’ strategy of homogeneous DNA detection.     

Significant enhancement of fluorescence on hybridization of a molecular beacon to a target DNA in the presence of a site-specific DNA nickase

We have developed a simple isothermal (55 degrees C) reaction that permits detection of DNA targets using only two components: a molecular beacon and a site-specific DNA nickase without deoxyribonucleotide triphosphates and primers. The loop sequence of the molecular beacon should contain a DNA nickase recognition site. The nickase-molecular beacon (NMB) combination permits a 100-fold increase in fluorescent signal. The applications of the NMB assay for enhancement of fluorescent signal in some isothermal methods are discussed.     

Synchrotron excitation of DNA fluorescence. Decay time evidence for excimer emission at room temperature

The first lifetime measurements of DNA fluorescence are reported. Natural and synthetic DNA have been excited by 1.76 ns pulses of synchrotron ultraviolet radiation (270 nm) and the time profile of the fluorescence has been measured by synchronous single-photon counting. A post-pulse exponentially decaying emission has been observed with a lifetime of 2.9 +/- 0.4 ns for calf thymus DNA and 3.0 +/- 0.3 ns for poly(dA-T); this is most likely an excimer fluorescence.     

Development of a chromosomal DNA probe for the laboratory diagnosis of aspergillosis

Chromosomal DNA was extracted from clinical isolates of Aspergillus fumigatus of human and animal origin using the protoplast lysate method. The probe was developed by the nick translation of the chromosomal DNA genome fraction with p³² as the radiolabel. Hybridization of the probe with endonuclease-cleaved DNA of the same species resulted in a pattern of recognition sites specific for the species. The latter was not seen in other species encountered in clinical specimens. Trials were carried out on sputum experimentally inoculated with the fungus where crude DNA was directly extracted, treated with the endonuclease and hybridized with the probe. The efficacy of the probe was as good with the crude as the purified DNA. The specificity of the probe was determined by testing it against single and mixed DNA populations extracted from different species of several fungal and bacterial genera isolated from and/or known to occur in clinical specimens of respiratory infection origin. The sensitivity of the probe was assessed by detecting a DNA concentration in the specimen equivalent to 3 C.F.U.This research project was supported by the Mayo Visiting Clinician Program, Mayo Medical Center, Rochester, MN 55905, USA.     

Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid

We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the order of MB>ATssMB>GCssMB>TaqMan. The fluorescence quantum yield measurements indicate that incomplete or partial enzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-fluorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting fluorophore can strongly affect the probe performance in real-time PCR.     

Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level

Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.     

Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.     

Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27

The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4 pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases.     

Development of a new doubly-labeled fluorescent ceramide probe for monitoring the metabolism of sphingolipids in living cells

A new ceramide analog, 1, containing two fluorescent dyes, NBD in the N-acyl part and KFL5 in the alkyl part, was synthesized. The fluorescence from both NBD and KFL5 was detected in living cells in a time-dependent manner. A multi-wavelength fluorescence detector was used to detect ceramide metabolites including sphingosine, sphingosine-1-phosphate, glucosylceramide, and sphingomyelin, which are connected to the fluorescent dyes, simultaneously in a single TLC plate.     

Development of a PNA probe for the detection of the toxic dinoflagellate Takayama pulchella

A peptide nucleic acid (PNA) probe was developed to detect the toxic dinoflagellate, Takayama pulchella TPXM, using fluorescent in situ hybridization (FISH) combined with epifluorescent microscopy and flow cytometry. The PNA probe was then used to analyze HAB samples from Xiamen Bay. The results indicated that the fluorescein phosphoramidite (FAM)-labeled probe (PNATP28S01) [Flu]-OO ATG CCA TCT CAA GA, entered the algal cells easily and bound to the target species specifically. High hybridization efficiency (nearly 100%) was observed. Detection by epifluorescence microscopy and flow cytometry gave comparable results. The fluorescence intensity of the PNA probe hybridized to T. pulchella cells was remarkably higher than that of two DNA probes used in this study and than the autofluorescence of the blank and negative control cells. In addition, the hybridization condition of the PNA probe was easier to control than DNA probes, and when applied to field-collected samples, the PNA probe showed higher binding efficiency to the target species than DNA probes. With the observed high specificity, binding efficiency, and detection signal intensity, the PNA probe will be useful for monitoring harmful algal blooms of T. pulchella.     

Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA

To test the capacity of poxviruses for added foreign DNA, a recombinant was constructed that contains 24 700 bp of bacteriophage λ DNA inserted within the vaccinia virus thymidine kinase (TK) gene. The recombinant is stable, infectious and replicates in tissue culture at the same rate and to the same titer as standard vaccinia virus. This size flexibility of the poxvirus genome and the lack of stringent packaging requirements are useful features for an infectious eukaryotic cloning vector.     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

Identification of amino acids important for target recognition by the DNA:m5C methyltransferase M.NgoPII by alanine-scanning mutagenesis of residues at the protein-DNA interface

DNA:m(5)C MTases comprise a catalytic domain with conserved residues of the active site and a strongly diverged TRD with variable residues involved in DNA recognition and binding. To date, crystal structures of 2 DNA:m(5)C MTases complexed with the substrate DNA have been obtained; however, for none of these enzymes has the importance of the whole set of DNA-binding residues been comprehensively studied. We built a comparative model of M.NgoPII, a close homologue and isomethylomer of M.HaeIII, and systematically analyzed the effect of alanine substitutions for the complete set of amino acid residues from its TRD predicted to be important for DNA binding and target recognition. Our data demonstrate that only 1 Arg residue is indispensable for the MTase activity in vivo and in vitro, and that mutations of only a few other residues cause significant reduction of the activity in vitro, with little effect on the activity in vivo. The identification of dispensable protein-DNA contacts in the wild-type MTase will serve as a platform for exhaustive combinatorial mutagenesis aimed at the design of new contacts, and thus construction of enzyme variants that retain the activity but exhibit potentially new substrate preferences.     

Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: Identification of novel mutations

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene.