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PubMed2003

Reconstruction of DNA sequencing by hybridization.

MOTIVATION: It is widely recognized that the hybridization process is prone to errors and that the future of DNA sequencing by hybridization is predicated on the ability to successfully cope with such errors. However, the occurrence of hybridization errors results in the computational difficulty of the reconstruction of DNA sequencing by hybridization. The reconstruction problem of DNA sequencing by hybridization with errors is a strongly NP-hard problem. So far the problem has not been solved well. RESULTS: In this paper, a new approach is presented to solve the reconstruction problem of DNA sequencing by hybridization, which realizes the computational part of the SBH experiment. The proposed algorithm accepts both the negative and positive errors. The computational experiments show that the algorithm behaves satisfactorily, especially for the case with k-tuple repetitions and positive errors.

Algorithms↗

PubMed1981

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

Animals↗

PubMed1999

Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels.

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.

Base Sequence↗

PubMed1996

[High-throughput of DNA sequencing by automated machines].

I describe recent advance of DNA sequencing technology including automated machines. The human genome project, particularly large-scale DNA sequencing, forces us to handle numbers of samples simultaneously and rapidly. This lead us to develop new technologies. I herein introduce current progress of sequencing technologies and automated machines.

Human Genome Project↗

PubMed1987

Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification.

By means of the polymerase chain reaction (PCR) technique, DNA sequences were amplified that flank the crossover sites of a characteristic chromosomal translocation for follicular lymphomas, t(14;18)(q32;q21). This technique permitted the detection of cells carrying the t(14;18) hybrid DNA sequences at a dilution of 1:100,000. The remission marrow and blood samples of a patient with follicular lymphoma and the t(14;18) failed to show any abnormality by morphological examination and conventional Southern blot analysis. However, the t(14;18) hybrid DNA sequences were detected by the PCR technique. Thus, this technique is a highly sensitive tool to detect minimal residual cells carrying the t(14;18) and has the potential to identify a subpopulation of patients with subclinical disease.

Base Sequence↗

PubMed2006

Inference of DNA sequences from mechanical unzipping: an ideal-case study.

The performances of Bayesian inference to predict the sequence of DNA molecules from fixed-force unzipping experiments are investigated. We show that the probability of misprediction decreases exponentially with the amount of collected data. The decay rate is calculated as a function of biochemical parameters (binding free energies), the sequence content, the applied force, the elastic properties of a DNA single strand, and time resolution.

Bacteriophage lambda↗

PubMedDate not supplied

[Periodic classification of DNA sequences recognized by restrictases: properties of symmetry and regularity].

A circular periodic map of short palindromic DNA sequences is constructed using the sequence homology and symmetry. It is applied to compare sequences recognised by class II restriction and modification enzymes with other similar DNA sequences. All known restriction sites have two strong properties: they are enriched by GC pairs, and clustered (purine-purine, pyrimidine-pyrimidine) bonds predominant upon alternating ones. The preference of AT/GC alternation is only slight. These properties were compared quantitatively with the help of suggested numerical methods among different groups of restriction enzymes. The map is applied for prediction of new specificities of restriction modification systems. Possible mechanism of DNA sequence recognition by these enzymes and their evolution are discussed.

Base Sequence↗

PubMed1981

Mapping the topography of DNA wrapped around gyrase by nucleolytic and chemical probing of complexes of unique DNA sequences.

Complexes between DNA gyrase and DNA fragments of unique sequences were used to probe the topography of the DNA with nucleases and dimethyl sulfate. The results indicate that the flanking regions, each 50 bp in size, of a 145--155 bp DNA segment resistant to staphylococcal nuclease contain groups of pancreatic DNAase I-susceptible sites that are spaced 10--11 nucleotides apart. Pairs of adjacent DNAase I-sensitive sites on complementary strands are typically staggered by 2--4 bp. The binding of DNA to gyrase confers no protection against alkylation of the DNA by dimethyl sulfate. These properties of the gyrase-DNA complex are reminiscent of those of the nucleosome, and the common underlying structural feature appears to be wrapping of the DNA around a protein core. The gyrase-DNA complex differs from the nucleosome, however, in that it must possess features necessary for the catalysis of DNA chain breakage and the modulation of the DNA-enzyme interaction by ATP. We present evidence that the breakage and rejoining of the DNA by gyrase occur within a central region of the staphylococcal nuclease-resistant DNA segment. The relation of this observation to the mechanism of DNA supercoiling by gyrase is discussed. Addition of ATP or its beta, gamma-imido analog has essentially no effect on the patterns of susceptibilities to DNAase I, implying that the DNA-enzyme contacts mapped by the nuclease ae little affected by ATP-induced conformational changes.

Adenosine Triphosphate↗

PubMed1998

Quantitation of mixed-base populations of HIV-1 variants by automated DNA sequencing with BODIPY dye-labeled primers.

Direct DNA sequencing of human immunodeficiency virus type 1 (HIV-1) pol and env gene regions was characterized for accuracy and precision. Restricted maximum likelihood (REML) analysis of molecular clone reconstruction experiments using a primer-walking strategy showed that the BODIPY and BODIPY energy-transfer (BET) dye primers sets were significantly more accurate in quantitating heterogenous base mixtures than the fluorescein/rhodamine dye primers. Of the three sets examined, the BET dye primers were the most accurate. The improved accuracy correlated with the reduced emission band-widths of BODIPY and BET dye primers and the more uniform signal intensities of BET dye primers. However, comparing % coefficients of variation (CV) for the three dye primer sets, revealed that BODIPY dye primers gave better precision than both BET and fluorescein/rhodamine dye primer sets. Several sequence-dependent motifs were identified that showed specific nucleotide-biased incorporation and were determined to be the major variable component of the total %CV. Taken together, these results show that BODIPY and BET direct DNA sequencing can accurately and precisely characterize complex mixed-base populations.

Boron Compounds↗

PubMed1984

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs.

We have examined aspects of the interaction of cycled microtubule protein preparations with 35S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained 35S-labelled mouse tracer DNA. Filter retention levels increased if 35S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the 35S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with 35S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895-908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of 35S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S1 nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm3) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm3) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777-784). That the high-affinity microtubule-bound DNA was some 3-5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern.

Animals↗

PubMed1987

Unusual DNA sequences located within the promoter region and the first intron of the chicken pro-alpha 1(I) collagen gene.

Genomic clones corresponding to the amino-terminal propeptide and 5'-flanking sequences of the chicken pro-alpha 1(I) collagen gene were isolated as a first step in the identification of DNA sequences important for transcriptional regulation of the pro-alpha 1(I) collagen gene. Due to the failure to identify positive clones in either primary or amplified genomic libraries, a 5.1-kilobase pair StuI genomic fragment identified by Southern blotting was enriched by sucrose gradient fractionation of genomic DNA and cloned into lambda gt11. Comparison of the DNA sequence of the 5.1-kilobase pair StuI fragment to the DNA sequence of a cDNA clone encoding the amino-terminal propeptide, signal peptide, and the 5'-untranslated region identified the first four exons and most of the fifth. Exon size and intron position have been largely conserved between human and chicken alpha 1(I) genes. DNA sequence analysis of the region 5' to the transcription initiation site identified the canonical TATA and CAAT boxes. However, the 40-nucleotide pyrimidine stretch centered between -150 and -180 nucleotides, found in all previously isolated type I procollagen genes from chicken, mouse, and human, was absent in the chicken pro-alpha 1(I) collagen gene. This sequence corresponds to the in vivo DNase I hypersensitive site in the chicken pro-alpha 2(I) and mouse pro-alpha 1(I) collagen genes, as well as the in vitro S1 nuclease hypersensitive site in both chicken and mouse pro-alpha 2(I) collagen genes. Two unusual DNA sequences were identified within the chicken pro-alpha 1(I) collagen gene. Fifteen tandem repeats of the sequence GGGGAGA were identified within the first intron, 300 nucleotides 3' to the first exon. This sequence was identified due to its hypersensitivity to S1 nuclease in vitro in supercoiled plasmids. The second sequence located 5' to -180 contained at least 25 copies of a polymorphic, 23-base pair tandemly repeated sequence not identified in other type I procollagen genes. Both of these tandem repeat sequences were identified at other locations in the chicken genome by Southern blot hybridization.

Amino Acid Sequence↗

PubMed1995

The short DNA sequences in the cytoplasm of Ehrlich ascites tumor cells are tightly associated with proteins.

Mouse Ehrlich tumor cells harbor extrachromosomal DNA elements in a non-mitochondrial fraction of the cytoplasm (Abken et al., Proc. Natl. Acad. Sci USA 90: 6518-6522, 1993). The cytoplasmic DNA sequences constitute a distinct group of extrachromosomal genetic elements with common properties: (i) the DNA molecules are 50-500 bp in length and of linear configuration, (ii) most of the DNA sequences exhibit the potential to modulate the activity of a transcriptional promoter, and (iii) the DNA elements are preferentially found in tumor cells, not in cells with normal phenotype. Unexpectedly, the extrachromosomal DNA sequences are found to be tightly associated with at least three proteins (52 kD, 62 kD, 64 kD) forming DNA-protein (DNP) complexes. The DNA-protein interaction is stable during extraction with phenol and chloroform and during incubation with proteinase K and pronase P, in the presence of detergents (SDS, NP40), guanidine-HCl, high salt concentrations, and alkali. Hydrolysis of the DNA by DNAse I makes the proteins of the DNP complex accessible to proteolytic degradation. Western blot analyses imply that the proteins associated with the cytoplasmic DNA sequences are antigenically related to nucleomatrix proteins that are covalently bound to certain regions of chromosomal DNA. Covalent bonds between the cytoplasmic DNA and the polypeptides would explain the unexpected high stability of the cytoplasmic DNA-protein complexes in tumor cells.

Animals↗

PubMed1998

Mapping and molecular characterization of five HMG1-related DNA sequences.

Recently, the high mobility group (HMG) proteins have attracted a lot of interest since it was shown that some members of that group can causally be involved in tumorigenesis. One HMG protein gene member is HMG1 for which the number of related DNA sequences has been estimated to be approximately 20-30. Nevertheless, besides the gene for HMG1 only one retropseudogene has been molecularly characterized. It was the aim of this study to map and characterize further sequences related to HMG1. PCR-screening of a PAC library resulted in 25 very strongly positive clones apparently containing HMG1-like cDNA sequences. Of eight clones which were further investigated five were distinguishable from each other based on their chromosome assignment and DNA sequence. Due to their homology to the HMG1 gene the DNA sequences were designated as HMG1L1, HMG1L3, HMG1L4, HMG1L5, and HMG1L6. By FISH experiments they were assigned to 2q32, 2q35, 3p24, 15q22, and 20q13, respectively. Except for one sequence, they did not show mutations leading to a frame shift or a new termination codon. Thus, we cannot exclude that these four HMG1-related DNA sequences represent active genes or can at least be activated e.g. by chromosome rearrangements in tumor cells. So far, the existence of six genes encoding HMG proteins has been described but because of a high frequency of closely related DNA sequences in the human genome it can be assumed that some of them are either pseudogenes or very similar genes.

Blotting, Southern↗

PubMed2005

Molecular recognition via triplex formation of mixed purine/pyrimidine DNA sequences using oligoTRIPs.

Stable DNA triple-helical structures are normally restricted to homopurine sequences. We have described a system of four heterocyclic bases (TRIPsides) that, when incorporated into oligomers (oligoTRIPs), can recognize and bind in the major groove to any native sequence of DNA [Li et al., J. Am. Chem. Soc. 2003, 125, 2084]. To date, we have reported on triplex-forming oligomers composed of two of these TRIPsides, i.e., antiTA and antiGC, and their ability to form intramolecular triplexes at mixed purine/pyrimidine sequences. In the present study, we describe the synthesis and characterization of the antiCG TRIPside and its use in conjunction with antiTA and antiGC to form sequence-specific intra- and/or intermolecular triplex structures at mixed purine/pyrimidine sequences that require as many as four major groove crossovers.

Base Sequence↗

PubMed2000

Changes in primary DNA sequence complexity influence the phenotypic consequences of mutations in human gene regulatory regions.

No general rules have been proposed to account for the functional consequences of gene regulatory mutations. In a first attempt to establish the nature of such rules, an analysis was performed of the DNA sequence context of 153 different single base-pair substitutions in the regulatory regions of 65 different human genes underlying inherited disease. Use of a recently proposed measure of DNA sequence complexity (taking into account the level of structural repetitiveness of a DNA sequence, rather than simply the oligonucleotide composition) has served to demonstrate that the concomitant change in local DNA sequence complexity surrounding a substituted nucleotide is related to the likelihood of a regulatory mutation coming to clinical attention. Mutations that led to an increase in complexity exhibited higher odds ratios in favour of pathological consequences than mutations that led to a decrease or left complexity unchanged. This relationship, however, was discernible only for pyrimidine-to-purine transversions. Odds ratios for other types of substitution were not found to be significantly associated with local changes in sequence complexity, even though a trend similar to that observed for Y-->R transversions was also apparent for transitions. These findings suggest that the maintenance of a defined level of DNA sequence complexity, or at least the avoidance of an increase in sequence complexity, is a critical prerequisite for the function of gene regulatory regions.

Base Sequence↗

PubMed1992

Partial-digest DNA sequencing.

A technique called partial-digest sequencing that permits DNA of 4-6 kb in length to be sequenced without subcloning is described. The method exploits the specific cuts introduced by partial digestion with restriction endonucleases that have 4-base recognition sites to produce ordered ladders of PCR-amplified fragments. The staggered ends contain PCR primers and can thus be individually sequenced using conventional methods to yield overlapping sequences covering the entire region. This method should have significant impact on both large and small DNA sequencing projects and find many applications in general manipulations in which ordered sets of deletions need to be produced.

Animals↗

PubMed1995

DMSO resolves certain compressions and signal dropouts in fluorescent dye labeled primer-based DNA sequencing reactions.

Automated base calling algorithms are more sensitive to the quality of the DNA sequencing data than are the labor intensive visual methods of base calling. To improve this quality, data from DNA sequencing reactions have been compared in order to determine the effects of the inclusion of dimethyl sulfoxide (DMSO). Inclusion of 10% DMSO into the reaction cocktail resolves at least one type of sequence compression. This compression may be due to the lack of ability in T7 DNA polymerase to read through certain sequences correctly. The poor quality of these data is seen as radioactive bands or fluorescent signal peaks that have an abnormal alignment, either in the wrong order or as single bands/peaks. The inclusion of DMSO also resolves sequences where the peak signal is absent or severely diminished, leading to a "gap" in the chromatogram profile. DMSO is better than deaza-dITP for resolving certain compressions. Addition of DMSO is a cheaper and more efficient method for high-throughput DNA sequencing than repeating reactions with base analogs.

Algorithms↗

PubMed2002

Highly repeated DNA sequences in three species of the genus Pteropus (Megachiroptera, Mammalia).

Bat genomes are characterised by an A-T richness and by a small C-value compared with other mammalian groups. It has been suggested that the small C-value is mainly due to the lack of repetitive DNA sequences. However, little information about repetitive DNA sequences in this mammalian group is available at the molecular level. Here we describe a PstI family of repetitive DNA sequences present in three species of the genus Pteropus. These repetitive sequences are 54.97% G-C rich, organised in tandem and with a unit length of 744 bp. Methylation analysis indicates that some of the CCGG target sites present in these repetitive DNA sequences have methylated cytosines and that there are small differences in the methylation pattern between species. Several features of this family of repetitive sequences suggest that they evolved by concerted evolution.

Animals↗