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At least 487 records · Page 27Linked to original sources
PubMed2001

Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment.

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.

Carbohydrate Conformation↗
PubMed1991

EcoRV restriction endonuclease binds all DNA sequences with equal affinity.

In the presence of MgCl2, the EcoRV restriction endonuclease cleaves its recognition sequence on DNA at least a million times more readily than any other sequence. In this study, the binding of the EcoRV restriction enzyme to DNA was examined in the absence of Mg2+. With each DNA fragment tested, several DNA-protein complexes were detected by electrophoresis through polyacrylamide. No differences were observed between isogenic DNA molecules that either contained or lacked the EcoRV recognition site. The number of complexes with each fragment varied with the length of the DNA. Three complexes were formed with a DNA molecule of 55 base pairs, corresponding to the DNA bound to 1, 2, or 3 molecules of the protein, while greater than 15 complexes were formed with a DNA of 381 base pairs. A new method was developed to analyze the binding of a protein to multiple sites on DNA. The method showed that the EcoRV enzyme binds to all DNA sequences, including the EcoRV recognition site, with the same equilibrium constant, though two molecules of the protein bind preferentially to adjacent sites on the DNA in a cooperative fashion. All of the complexes with a substrate that contained the EcoRV site dissociated upon addition of competitor DNA, but when the competitor was mixed with MgCl2, a fraction of the substrate was cleaved at the EcoRV site. The fraction cleaved was due mainly to the translocation of the enzyme from nonspecific sites on the DNA to the specific site.

Binding, Competitive↗
PubMed1989

A system for on-line detection and resolution of radiolabeled DNA molecules and its application to automated DNA sequence analysis.

We describe a system for the real-time detection of radioactively labeled DNA molecules in gel matrix, and we demonstrate the application of this system to DNA sequence analysis. DNA sequencing reactions prepared by the Sanger chain termination method are resolved by electrophoresis on 8% polyacrylamide gels. During electrophoresis the 32P-labeled DNA fragments are detected by solid state detectors positioned 22 cm from the top surface of the gel. This system is able to resolve a DNA sequence of 300 bases or greater. Optimized protocols that allow sequence information to be obtained from single stranded and double-stranded templates are described. A linear relationship exists between the input dpm and the integrated peak values over a 20-fold range indicating that accurate DNA quantitation is also possible using this system.

Automation↗
PubMed1997

Laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS) to detect and characterise HPV DNA sequences by FISH in HeLa cells.

Visualisation and localisation of specific DNA sequences were performed by fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy (CLSM), and factor analysis of biomedical image sequences (FAMIS). HeLa cells containing 10-50 copies per cell of human papillomavirus (HPV) DNA type 18 integrated in cellular DNA were used as a model. HPV-DNA was identified by DNA probes and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red (FR) TR salt/naphtol-MX phosphate. Cell nuclei were counterstained with thiazole orange (TO). FAMIS summarises image sequences into a reduced number of images called factor images and curves called factors. Factor images correspond to spatial distributions of the different factors. Factors estimate different individual physical behaviours in the sequence (extinction velocity, spectral emission, depth emission profiles). We verified that HPV-DNA hybridisation signals are specific to the spectrum of FR, and distinguished between FR and TO. The latter result was found by taking into account differences in their extinction velocities. The focus of CLSM was improved on 3D image sequences, and the location of fluorescent signals inside the preparations was determined. Factor images showed that FR stained targets were located on different focal planes at the periphery of the nuclei.

DNA, Viral↗
PubMed1984

A new procedure for determining thymine residues in DNA sequencing. Photoinduced cleavage of DNA fragments in the presence of spermine.

A new procedure for T specific cleavage of DNA fragments utilizing photoreaction with spermine has been described. Irradiation of 3'-[32P]-end-labeled DNA fragments for 10-20 min with a germicidal lamp emitting mainly 254-nm light in the presence of 1 M spermine in distilled water resulted in a T specific cleavage of the DNA chains. This method does not require piperidine treatment. By contrast, when the DNA fragments were irradiated in the presence of methylamine under similar conditions, both G and T bands with the intensity of G greater than T have appeared. A similar but less selective T cleavage has also been observed in the irradiation of 5'-[32P]-end-labeled DNA fragments in the presence of spermine followed by brief heating of the photolysate in a loading buffer for gel electrophoresis. The T specific photoreaction with spermine and the G greater than T reaction with methylamine described here may be conveniently used in combination with the standard Maxam-Gilbert's reactions to provide independent confirmatory readings.

Autoradiography↗
PubMed2003

DNA sequence representation without degeneracy.

Graphical representation of DNA sequence provides a simple way of viewing, sorting and comparing various gene structures. A new two-dimensional graphical representation method using a two- quadrant Cartesian coordinates system has been derived for mathematical denotation of DNA sequence. The two-dimensional graphic representation resolves sequences' degeneracy and is mathematically proven to eliminate circuit formation. Given x-projection and y-projection of any point on the graphical representation, the number of A, G, C and T from the beginning of the sequence to that point could be found. Compared with previous methods, this graphical representation is more in-line with the conventional recognition of linear sequences by molecular biologists, and also provides a metaphor in two dimensions for local and global DNA sequence comparison.

Animals↗
PubMed2004

Array-based comparative genomic hybridization for the detection of DNA sequence copy number changes in Barrett's adenocarcinoma.

Array-based comparative genomic hybridization (aCGH) allows the identification of DNA sequence copy number changes at high resolution by co-hybridizing differentially labelled test and control DNAs to a micro-array of genomic clones. The present study has analysed a series of 23 formalin-fixed, paraffin wax-embedded tissue samples of Barrett's adenocarcinoma (BCA, n = 18) and non-neoplastic squamous oesophageal (n = 2) and gastric cardia mucosa (n = 3) by aCGH. The micro-arrays used contained 287 genomic targets covering oncogenes, tumour suppressor genes, and DNA sequences localized within chromosomal regions previously reported to be altered in BCA. DNA sequence copy number changes for a panel of approximately 50 genes were identified, most of which have not been previously described in BCA. DNA sequence copy number gains (mean 41 +/- 25/BCA) were more frequent than DNA sequence copy number losses (mean 20 +/- 15/BCA). The highest frequencies for DNA sequence copy number gains were detected for SNRPN (61%); GNLY (44%); NME1 (44%); DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, and TNFRSF6B (39% each); and MSH2, TERC, SERPINE1, AFM137XA11, IGF1R, and PTPN1 (33% each). DNA sequence copy number losses were identified for PDGFB (44%); D17S125 (39%); AKT3 (28%); and RASSFI, FHIT, CDKN2A (p16), and SAS (CDK4) (28% each). In all non-neoplastic tissue samples of squamous oesophageal and gastric cardia mucosa, the measured mean ratios were 1.00 (squamous oesophageal mucosa) or 1.01 (gastric mucosa), indicating that no DNA sequence copy number chances were present. For validation, the DNA sequence copy number changes of selected clones (SNRPN, CMYC, HER2, ZNF217) detected by aCGH were confirmed by fluorescence in situ hybridization (FISH). These data show the sensitivity of aCGH for the identification of DNA sequence copy number changes at high resolution in BCA. The newly identified genes may include so far unknown biomarkers in BCA and are therefore a starting point for further studies elucidating their possible role in Barrett's carcinogenesis.

Adenocarcinoma↗
PubMed1997

An iterative and regenerative method for DNA sequencing.

This paper presents, to our knowledge, the first iterative DNA sequencing method that regenerates the product of interest during each iterative cycle, allowing it to overcome the critical obstacles that impede alternative iterative approaches to DNA sequencing: loss of product and the accumulation of background signal due to incomplete reactions. It can sequence numerous double-stranded (ds) DNA segments in parallel without gel resolution of DNA fragments and can sequence DNA that is almost entirely double-stranded, preventing the secondary structures that impede sequencing by hybridization. This method uses ligation of an adaptor containing the recognition domain for a class-IIS restriction endonuclease and digestion with a class-IIS restriction endonuclease that recognizes the adaptor's recognition domain. This generates a set of DNA templates that are each composed of a short overhang positioned at a fixed interval with respect to one end of the original dsDNA fragment. Adaptor ligation also appends a unique sequence during each iterative cycle, so that the polymerase chain reaction can be used to regenerate the desired template-precursor before class-IIS restriction endonuclease digestion. Following class-IIS restriction endonuclease digestion, sequencing of a nucleotide in each overhang occurs by template-directed ligation during adaptor ligation or through a separate template-directed polymerization step with labeled ddNTPs. DNA sequencing occurs in strides determined by the number of nucleotides separating the recognition and cleavage domains for the class-IIS restriction endonuclease encoded in the ligated adaptor, maximizing the span of DNA sequenced for a given number of iterative cycles. This method allows the concurrent sequencing of numerous dsDNA segments in a microplate format, and in the future it can be adapted to biochip format.

Base Sequence↗
PubMed1987

Preferential clustering of viral DNA sequences at or near the site of chromosomal rearrangement in fowl adenovirus type 1 DNA-transformed cell lines.

All six transformants obtained by inoculating fowl adenovirus type 1 (CELO virus) DNA or its fragments into a rat cell line of normal karyotype had more than 50 copy-equivalents of viral DNA sequences. Each of the transformants had almost all if not all of these viral DNA sequences clustered on a marker chromosome(s). Although the marker chromosome(s) differed from one cell line to another, viral DNA sequences preferentially clustered in or near the achromatic (or light-stained) region of the G-banded marker chromosomes where chromosomal rearrangement or translocation occurred. These results indicate that no particular chromosome is required to act as the integration site of viral DNA for the transformation of cells, but chromosomal rearrangement at or near the cluster of viral DNA sequences might contribute to the transformation.

Adenoviridae↗
PubMed1998

Short-sequence DNA repeats in prokaryotic genomes.

Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10(-4) per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant.

Adhesins, Bacterial↗
PubMed2005

Matrix conditioning for lengthened capillary DNA sequencing.

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.

DNA↗
PubMed1986

Rates of DNA sequence evolution differ between taxonomic groups.

The mutation rates of DNA sequences during evolution can be estimated from interspecies DNA sequence differences by assaying changes that have little or no effect on the phenotype (neutral mutations). Examination of available measurements shows that rates of DNA change of different phylogenetic groups differ by a factor of 5. The slowest rates are observed for higher primates and some bird lineages, while faster rates are seen in rodents, sea urchins, and drosophila. The rate of DNA sequence change has decreased markedly during primate evolution. The contrast in rates of DNA sequence change is probably due to evolutionary variation and selection of biochemical mechanisms such as DNA replication or repair.

Animals↗
PubMed2001

Random mutagenesis-PCR to introduce alterations into defined DNA sequences for validation of SNP and mutation detection methods.

Sensitive and high throughput techniques are required for the detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs) and mutations. One problem, common to all methods of SNP and mutation detection, is that experimental conditions required for detection of DNA sequence variants depend on the specific DNA sequence to be analyzed. Although algorithms and other calculations have been developed to predict the experimental conditions required to detect DNA sequence variation in a specific DNA sequence, these algorithms do not always provide reliable information and experimental conditions for SNP and mutation detection must be devised empirically. Determination of experimental conditions for detection of DNA sequence variation is difficult when samples containing only wild type sequence are available. When patient derived positive controls are used, increasingly there are valid concerns about commercial ownership and patient privacy. This report presents a rapid and efficient method, employing random mutagenesis-PCR (RM-PCR) using low fidelity DNA polymerase, to randomly introduce single and multiple base substitutions and deletions into DNA sequences of interest. Clones with sequence changes were used to validate denaturing HPLC (DHPLC) algorithm predictions, optimize conditions for mutation detection in exon 15 of the tyrosine kinase domain of the MET proto-oncogene, and to confirm the association between specific DNA sequence changes and unique DHPLC chromatographic profiles (signatures). Finally, DNA from 33 papillary renal carcinoma (PRC) patients was screened for mutations in exon 15 of MET using "validated" DHPLC conditions as a proof of principle application of RM-PCR. Use of RM-PCR for DHPLC and other SNP/mutation detection methods is discussed along with challenges associated with detecting sequence alterations in mixed tumor/normal tissue, pooled samples, and from regions of the genome that have been amplified during tumorigenesis or duplicated during evolution. Hum Mutat 17:210-219, 2001. Published 2001 Wiley-Liss, Inc.

Chromatography, High Pressure Liquid↗
PubMed1979

DNA sequencing and melting curve.

The dependence of DNA absorbance (for light at about 260 nm) on temperature is related to a specific DNA sequence structure in the vicinity of DNA thermal denaturation (the so-called DNA melting or coiling). A straightforward analysis of the experimental DNA melting curve allows us to determine the lengths, the A+T content, and the location in DNA of certain domains. In the case of a specific DNA fragmentation, the order of fragments in DNA can be learned from this analysis, nondestructively and quickly, without fractionating the fragments and other methods of fragmentation. If the DNA nucleotide sequence is known except for some sites and uncertain portions, the analysis determines these sites and the accuracy of the sequence at the portions. This information may complement exact methods of DNA sequencing. The proposed analysis is applied to bacteriophage phiX174, whose melting curve is known. The results are compared to and found to be in an excellent agreement with the known phiX174 nucleotide sequence.

Base Sequence↗
PubMed2003

Multiple-primer DNA sequencing method.

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.

DNA Primers↗
PubMed1994

DNA sequences specific for Caucasian G3m(b) and (g) allotypes: allotyping at the genomic level.

Assignment of the G3m(g) and (b) correlative amino acid residues was performed at the genomic level by direct sequencing of DNA from nine Caucasian individuals. Two oligonucleotide primers were used for subclass-specific enzymatic amplification of a DNA segment comprising a major portion of the second and third constant region domains (CH2 and CH3) of the human IgG3 heavy chain gene. Comparison of the sequences of amplified DNA from individuals serologically typed as homozygous for G3m(b) or G3m(g) or as heterozygous, G3m(b,g), revealed differences in the codons for the amino acid residues 291, 296, and 384. Proline, phenylalanine, and serine at these positions corresponded to G3m(b), and leucine, tyrosine, and asparagine to G3m(g). Heterozygotic individuals, typed G3m(b,g), displayed both the G3m(b) and G3m(g) codons at these three positions. The polymorphism at each of these three codons could be identified either as the appearance, or the loss, of recognition sites for the two restriction endonucleases, Nsp BII and Rsa I. This allowed the development of a polymerase chain reaction (PCR)-based assay permitting the distinction of G3mb and G3mg alleles by analyzing the electrophoretical mobility of the DNA fragments generated by digestion of the PCR-products with Nsp BII and Rsa I.

Amino Acid Sequence↗
PubMed2002

Finding composite regulatory patterns in DNA sequences.

Pattern discovery in unaligned DNA sequences is a fundamental problem in computational biology with important applications in finding regulatory signals. Current approaches to pattern discovery focus on monad patterns that correspond to relatively short contiguous strings. However, many of the actual regulatory signals are composite patterns that are groups of monad patterns that occur near each other. A difficulty in discovering composite patterns is that one or both of the component monad patterns in the group may be 'too weak'. Since the traditional monad-based motif finding algorithms usually output one (or a few) high scoring patterns, they often fail to find composite regulatory signals consisting of weak monad parts. In this paper, we present a MITRA (MIsmatch TRee Algorithm) approach for discovering composite signals. We demonstrate that MITRA performs well for both monad and composite patterns by presenting experiments over biological and synthetic data.

Algorithms↗