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At least 163 records · Page 9Linked to original sources
PubMed1992

Photodestruction of fluorophores and optimum conditions for trace DNA detection by automated DNA sequencer.

Although automated DNA sequencers are becoming popular, their sensitivity in detecting DNA bands is still around 10(-17) mole/band. The sensitivity of a system depends on the laser power, labeling fluorophore, and the fluorescence-collecting yield. The emission and photodestruction cross-sections of the fluorophores are critical in optimizing the irradiated laser power and the migration speeds of DNA fragments to achieve high sensitivity. We investigated photodestruction cross-sections of various fluorophores to optimize the irradiation laser power. In addition, we used a cylindrical lens system to improve the fluorescence-collecting yield of a DNA sequencer using side entry laser irradiation. Fluoresceine isothiocyanate (FITC) commonly used in fluorescence studies, is very photo-destructive, the cross-section of the destruction being about 3.8 x 10(-20) cm2 in buffer solution while that of Texas Red is 1.5 x 10(-21) cm2. When the time for DNA fragments to transit through the irradiated region is 11 s, the optimum laser powers are 0.9 mW, with an Ar laser (488 nm) for FITC-DNA, and 18 mW, with an He-Ne laser (594 nm) for Texas Red DNA. We have developed a DNA sequencer, with a cylindrical lens system which improves the fluorescence-collecting efficiency by a factor of 4, and an He-Ne laser (5 mW). Although the sequencer uses a slab gel, an ultra-high sensitivity of 5 x 10(-20) mole/band (S/N-4) was achieved under optimized conditions.

Autoanalysis↗
PubMed1985

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.

Animals↗
PubMed1996

Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes.

We describe a rapid method for visually determining specific DNA sequences at femtomole concentrations. Liposomes, encapsulating a red dye and labeled with oligonucleotide, were used in a capillary migration-sandwich hybridization assay. Capture probe was immobilized on nitrocellulose strips, and liposomes, migrating along each strip, formed a visually discernible band in the presence of target DNA. One femtomole of synthetic target sequence could be detected in < 10 min. Sufficiently stringent hybridization conditions can be used to allow the discrimination of a 10% mismatch sequence from perfectly complementary DNA. A 366-base PCR product was detected at 200 fmol.

Base Sequence↗
PubMed2004

The ribosomal RNA gene promoter and adjacent cis-acting DNA sequences govern plasmid DNA partitioning and stable inheritance in the parasitic protozoan Leishmania.

Detailed analysis of the Leishmania donovani ribosomal RNA (rRNA) gene promoter region has allowed the identification of cis-acting sequences involved in plasmid DNA partitioning and stable plasmid inheritance. We report that plasmids bearing the 350 bp rRNA promoter along with the 200 bp region immediately 3' to the promoter exhibited a 6.5-fold increase in transformation frequency and were transmitted to daughter cells as single-copy molecules. This is in contrast to what has been observed for plasmid molecules in this organism so far. Moreover, we show that these low-copy-number plasmids displayed a remarkable mitotic stability in the absence of selective pressure. The region in the vicinity of the RNA pol I transcription initiation site, and also in the adjacent 200 nt, displays a complex structural organization and shares sequence similarity to the yeast autonomously replicating consensus sequence and centromere DNA elements. Deletion analyses indicated that these elements were necessary but not sufficient for plasmid DNA partitioning and stable inheritance, and that the rRNA promoter region was required for optimal function. These results suggest an interplay between RNA pol I transcription, DNA replication, DNA partitioning and mitotic stability in trypanosomatids. This is the first example of defined DNA elements for plasmid partitioning and stable inheritance in the protozoan parasite Leishmania.

Animals↗
PubMed2006

[A new method for visual SARS DNA sequences analysis].

Traditional DNA sequence analysis is based on sequence alignment, while a new DNA visual sequence analysis is proposed in this paper. Based on S. Wolfram's cellular automation theory, the method transfers one-dimensional DNA sequence into two-demensional visual image. Applying this method to SARS DNA sequence analysis, a characteristic of SARS-CoV differing from non-SARS is discovered. Compared with all known coronaviruses' images, It is found that this is a unique characteristic of SARS virus, and it is helpful to clinical identification of SARS.

Algorithms↗
PubMed1999

Intranuclear anchoring of repetitive DNA sequences: centromeres, telomeres, and ribosomal DNA.

Centromeres, telomeres, and ribosomal gene clusters consist of repetitive DNA sequences. To assess their contributions to the spatial organization of the interphase genome, their interactions with the nucleoskeleton were examined in quiescent and activated human lymphocytes. The nucleoskeletons were prepared using "physiological" conditions. The resulting structures were probed for specific DNA sequences of centromeres, telomeres, and ribosomal genes by in situ hybridization; the electroeluted DNA fractions were examined by blot hybridization. In both nonstimulated and stimulated lymphocytes, centromeric alpha-satellite repeats were almost exclusively found in the eluted fraction, while telomeric sequences remained attached to the nucleoskeleton. Ribosomal genes showed a transcription-dependent attachment pattern: in unstimulated lymphocytes, transcriptionally inactive ribosomal genes located outside the nucleolus were eluted completely. When comparing transcription unit and intergenic spacer, significantly more of the intergenic spacer was removed. In activated lymphocytes, considerable but similar amounts of both rDNA fragments were eluted. The results demonstrate that: (a) the various repetitive DNA sequences differ significantly in their intranuclear anchoring, (b) telomeric rather than centromeric DNA sequences form stable attachments to the nucleoskeleton, and (c) different attachment mechanisms might be responsible for the interaction of ribosomal genes with the nucleoskeleton.

Cell Nucleus↗
PubMed1986

Analysis of DNA sequences which regulate the transcription of herpes simplex virus immediate early gene 3: DNA sequences required for enhancer-like activity and response to trans-activation by a virion polypeptide.

The far upstream region of herpes simplex virus (HSV) immediate early (IE) gene 3 has previously been shown to increase gene expression in an enhancer-like manner, and to contain sequences which respond to stimulation of transcription by a virion polypeptide, Vmw65. To analyse the specific DNA sequences which mediate these functions, sequential deletions from each end of the far upstream region were made. The effects of the deletions on transcription in the absence or presence of the Vmw65 were measured by use of a transient expression assay. The enhancer-like activity was due to three separable elements, whereas two additional DNA regions were involved in the response to Vmw65. One of the responding elements corresponded to an AT-rich consensus (TAATGARATTC, where R = purine) present in all IE gene far upstream regions, and the other was a GA-rich sequence also present in IE genes 2 and 4/5. The TAATGARATTC element could mediate responsiveness to Vmw65 but it was fully active only in the presence of the GA-rich element. The GA-rich element was unable to confer a strong response alone but could activate an otherwise nonfunctional homologue of TAATGARATTC.

Animals↗
PubMed1997

The study of correlation structures of DNA sequences: a critical review.

The study of correlation structure in the primary sequences of DNA is reviewed. The issues reviewed include: symmetries among 16 base-base correlation functions; accurate estimation of correlation measures; the relationship between 1/f and Lorentzian spectra; heterogeneity in DNA sequences; different modeling strategies of the correlation structure of DNA sequences; the difference of correlation structure between coding and non-coding regions (besides the period-3 pattern); and source of broad distribution of domain sizes. Although some of the results remain controversial, a body of work on this topic constitutes a good starting point for future studies.

Base Sequence↗
PubMed1987

Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP.

It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site. We have purified wild-type CAP and two substituted CAP variants, [Val181]CAP and [Leu181]CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site. The data indicate that [Val181]CAP and [Leu181]CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site. In contrast, [Val181]CAP and [Leu181]CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site. We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results. We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact. We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.

Amino Acid Sequence↗
PubMed2003

A photocleavable fluorescent nucleotide for DNA sequencing and analysis.

DNA sequencing by synthesis during a polymerase reaction using laser-induced fluorescence detection is an approach that has a great potential to increase the throughput and data quality of DNA sequencing. We report the design and synthesis of a photocleavable fluorescent nucleoside triphosphate, one of the essential molecules required for the sequencing-by-synthesis approach. We synthesized this nucleoside triphosphate by attaching a fluorophore, 4,4-difluoro-5,7-dimethyl-4-bora-3alpha,4alpha-diaza-s-indacene propionic acid (BODIPY), to the 5 position of 2'-deoxyuridine triphosphate via a photocleavable 2-nitrobenzyl linker. We demonstrate that the nucleotide analogue can be faithfully incorporated by a DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA-sequencing reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. These results indicate that the nucleotide analogue is an excellent substrate for Thermo Sequenase. We also systematically studied the photocleavage of the fluorescent dye from a DNA molecule that contained the nucleotide analogue. UV irradiation at 340 nm of the DNA molecule led to the efficient release of the fluorescent dye, ensuring that a previous fluorescence signal did not leave any residue that could interfere with the detection of the next nucleotide. Thus, our results indicate that it should be feasible to use four different fluorescent dyes with distinct fluorescence emissions as unique tags to label the four nucleotides (A, C, G, and T) through the photocleavable 2-nitrobenzyl linker. These fluorescent tags can be removed easily by photocleavage after the identification of each nucleotide in the DNA sequencing-by-synthesis approach.

Biotin↗
PubMed1982

Representation of DNA sequences in recombinant DNA libraries prepared by restriction enzyme partial digestion.

We present a theoretical study of the fraction of sequences incorporated in a recombinant DNA partial digest library as a function of the size of the library. The fraction incorporated depends on the degree of restriction enzyme partial digestion. If all restriction sites in the target DNA can be cleaved with the same rate, optimum incorporation of sequences is observed when the number average length of the digested DNA equals the desired average length of the cloned insert. Overdigestion severely reduces the fraction of sequences present in a sample of clones. Heterogeneity in restriction enzyme cleavage rates also reduces the fraction incorporated, and underdigestion improves sequence representation in the face of cleavage rate heterogeneity. Practical methods for determining the number average length of partially digested DNAs are also presented.

Base Composition↗
PubMed1991

'Size leap' algorithm: an efficient extraction of the longest common motifs from a molecular sequence set. Application to the DNA sequence reconstruction.

We propose a new method, called 'size leap' algorithm, of search for motifs of maximum size and common to two fragments at least. It allows the creation of a reduced database of motifs from a set of sequences whose size obeys the series of Fibonacci numbers. The convenience lies in the efficiency of the motif extraction. It can be applied in the establishment of overlap regions for DNA sequence reconstruction and multiple alignment of biological sequences. The method of complete DNA sequence reconstruction by extraction of the longest motifs ('anchor motifs') is presented as an application of the size leap algorithm. The details of a reconstruction from three sequenced fragments are given as an example.

Algorithms↗
PubMed1989

In vitro transcription analysis of the role of flanking sequence on the DNA sequence specificity of adriamycin.

An in vitro transcription assay has been used to define twelve high occupancy transcriptional blockage sites in 260 bp of heterogenous DNA on three promoter-containing DNA fragments. Transcription proceeded to immediately upstream of CpA sequences in nine of these sites, and this defines the most preferred intercalation site as CpA. In almost all cases, this sequence was flanked by T on the 5' end. The consensus sequence for the highest affinity Adriamycin site is therefore 5'-TCA, with some evidence for preference of AT base pairs flanking both ends of this trinucleotide [i.e., (t)TCA(a.t)(a.t)]. In contrast, lower occupancy CpA sites were flanked on the 5' end by a GC base pair, with a preference for up to three GC base pairs on the 3' end. The low affinity consensus sequence is therefore (G.C)CA(g.c)(g.c)(g.c).

Base Sequence↗
PubMed1990

Rapid detection and sequencing of specific in vitro amplified DNA sequences using solid phase methods.

We describe a rapid solid phase assay for detection and sequencing of DNA sequences based on selective introduction of biotin and isotope into the specific DNA fragment amplified by the polymerase chain reaction (PCR). A two-step PCR procedure is used to lower the background signal. The in vitro amplified material is immobilized on magnetic beads with covalently coupled streptavidin and the amount of bound label is measured. Samples identified as positive can be analysed by direct solid phase DNA sequencing. A strategy is also described to use general primers for detection, capturing and sequencing, which are not homologous to the specific sequence to be detected. The concept has been optimized using oligonucleotides specific for Staphylococci and Streptococci, respectively. Here, we show that the assay can be used for detection of Plasmodium falciparum in clinical samples.

Animals↗
PubMed1988

Effect of base composition at the center of inverted repeated DNA sequences on cruciform transitions in DNA.

We have analyzed the effect of base composition at the center of symmetry of inverted repeated DNA sequences on cruciform transitions in supercoiled DNA. For this we have constructed two series of palindromic DNA sequences: one set with differing center and one set with differing center and arm sequences. The F series consists of two 96-base pair perfect inverted repeats which are identical except for the central 10 base pairs which consist of pure AT or GC base pairs. The S series was constructed such that the overall base composition of the inverted repeats was identical but in which the positioning of blocks of AT- and GC-rich sequences varied. The rate of cruciform formation for the inverted repeats in plasmid pUC8 was dramatically influenced by the 8-10 base pairs at the center of the inverted repeat. Inverted repeats with 8-10 AT base pairs in the center were kinetically much more active in cruciform formation than inverted repeats with 8-10 GC base pairs in the center. These experiments show a dominant influence of the center sequences of inverted repeats on the rate of cruciform formation.

Base Sequence↗
PubMed1977

Chromatin transcription. I. Quantitative determination of gene-specific RNA by use of bacterial plasmids containing the eukaryotic DNA sequence of viral DNA.

Specific gene transcription from chromatin has been examined in two different systems by using direct measurement of specific sequences by hybridization with excess DNA probe. An improved procedure has been used for hybridization of DNA immobilized on filters. This procedure is sensitive to less than one part in 10(5). Polyoma RNA sequences were detected a level of 0.004% in the overall transcript from chromatin of ts-a-transformed 3T3 fibroblasts. By the use of constructed plasmid DNA carrying rabbit globin sequences, globin RNA was titrated in the RNA transcribed from chromatin of bone marrow cells. Its relative frequency was 0.01%. Results obtained with this new approach confirm that DNA in chromatin is not randomly transcribed. They further illustrate that a reproducible assay system is now available for studying control elements active on gene expression at the transcriptional level, taking advantage of the constructed plasmids containing eukaryotic gene DNA fragments.

Animals↗
PubMed1992

Sequence accuracy of large DNA sequencing projects.

Very little information has been accumulated regarding the likely accuracy of final or consensus DNA sequence data. With the large-scale efforts anticipated for the Human Genome Project, the subjective determination of final sequence must eventually be replaced with more objective, automatic methods. This will require a much better understanding of the nature of error in raw sequencing data and its impact on the determination of the final sequence. This paper describes a start at defining the error model of large-scale sequencing efforts based on random subcloning strategies.

Consensus Sequence↗