carbocyanines and deoxyuridine-triphosphate

To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3- and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3' ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates.

Topics: Carbocyanines; Deoxyuracil Nucleotides; DNA-Directed DNA Polymerase; Fluorescent Dyes; Kinetics; Nucleotides; Nucleotidyltransferases; Polymerase Chain Reaction; Taq Polymerase; Templates, Genetic

Discrete chromatin domains (ChrD), containing an average of approximately 1 Mbp DNA, represent the basic structural units for the regulation of DNA organization and replication in situ. In this study, a bio-computational approach is employed to simultaneously measure the translational motion of large populations of ChrD in the cell nucleus of living cells. Both movement and configurational changes are strikingly higher in early S-phase replicating ChrD compared to those that replicate in mid and late S-phase. The chromatin dynamics was not sensitive to transcription inhibition by alpha-amanitin but was significantly reduced by actinomycin D treatment. Since a majority of active genes replicate in early S-phase, our results suggest a correlation between levels of chromatin dynamics and chromatin poised for active transcription. Analysis of ChrD colocalization with transcription sites and cDNA with ChrD and transcription sites further supports this proposal.

Topics: Alpha-Amanitin; Carbocyanines; Cell Nucleus; Chromatin; Chromosome Positioning; Dactinomycin; Deoxyuracil Nucleotides; DNA Replication; Gene Expression; HeLa Cells; Humans; In Situ Hybridization, Fluorescence; Kinetics; Microinjections; Microscopy, Fluorescence; S Phase; Time Factors

Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.

Topics: Base Sequence; Carbocyanines; Deoxyuracil Nucleotides; DNA Primers; DNA, Viral; Genotype; HeLa Cells; Humans; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Papillomaviridae; Papillomavirus Infections; Polymerase Chain Reaction; Sensitivity and Specificity; Sequence Alignment; Vaginal Smears

We present results of an approach in which low-density labeled DNA itself provides an amplification of the cross-correlated fluorescent signal in the two-color cross-correlation function. Tetramethylrhodamine-4-dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp target DNA. We call this novel approach the 'two-color FCS signal amplification'. The signal amplification is an example for interactions of two ligands with different colors at multiple positions of the same target.

Topics: Carbocyanines; Deoxyuracil Nucleotides; DNA; Fluorescent Dyes; Models, Theoretical; Polymerase Chain Reaction; Rhodamines; Spectrometry, Fluorescence

Synthesis of fluorochrome-modified deoxyribonucleotides has been carried out mostly by linking the fluorochrome molecule to the C-5 position of dUTP via an allylamine spacer, similar to the modification of allylamine-dUTP with the haptens biotin and digoxigenin. Recently, a new series of fluorescent nucleotides has been prepared by using an alkynyl bridge between the uracil moiety and the fluorochrome. Here we report the qualitative and quantitative analysis of fluorescence in situ hybridization results obtained on interphase cells and chromosomes with a variety of highly repetitive and single-copy DNA probes that were modified by nick translation with such alkynyl dUTPs. A qualitative comparison was made of the alkynyl dUTPs conjugated to the fluorochromes fluorescein, the cyanine dye Cy3, tetramethylrhodamine, Lissamine and Texas Red. With the exception of tetramethylrhodamine, all fluorochromes performed satisfactorily. The cyanine dye Cy3 provided the highest sensitivity, i.e., cosmid and YAC probes could easily be visualized by conventional fluorescence microscopy. In a quantitative assay, different nick translation conditions were tested using a human chromosome 1 satellite III probe (pUC1.77) and alkynyl dUTPs labeled with fluorescein and Cy3. Using these two nucleotides, FISH signal intensities on interphase nuclei from human lymphocytes were quantitated by digital imaging microscopy. The strongest signals were obtained when during nick translation the ratio between dTTP and fluorescein-dUTP or Cy3-dUTP was 1:5.

Topics: Carbocyanines; Chromosomes, Artificial, Yeast; Chromosomes, Human, Pair 1; Cosmids; Deoxyuracil Nucleotides; DNA Probes; Evaluation Studies as Topic; Fluorescein; Fluoresceins; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Leukocytes; Sensitivity and Specificity

Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR.

Topics: Carbocyanines; Deoxyuracil Nucleotides; DNA Probes; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Nucleic Acid Hybridization; Polymerase Chain Reaction