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At least 883 records · Page 49Linked to original sources
PubMed1984

On the statistical assessment of similarities in DNA sequences.

The statistical behavior of the similarity score for unrelated DNA sequences calculated as letter-by-letter comparison or from various forms of optimal alignment was studied. It was found that natural DNA-sequences from a data base and true random sequences show the same statistical behavior in terms of such scores. This makes it possible to adopt a simple criterion for the rejection of fortuitous similarity. It is based on the mean and standard deviation of chance scores whose expected values, depending on chain length, gap penalty and probability of letter coincidence, may be calculated from formulae given in the paper.

Base Sequence↗
PubMed1987

The DNA sequence specificity of stimulation of DNA polymerases by factor D.

The mechanism of enhancement of DNA polymerase activity by the murine DNA-binding protein factor D was investigated. Extension by Escherichia coli DNA polymerase I and calf thymus DNA polymerase-alpha of 5'-32P-labeled oligodeoxynucleotide primers that are complementary to poly(dT) or to bacteriophage M13 DNA was measured in the absence or presence of factor D. With 5'-[32P](dA)9.poly(dT), factor D enables E. coli polymerase I to fill approximately 15-nucleotide gaps between adjacent primers; whereas in the absence of the stimulatory protein, poly(dT) is not copied significantly. In order to study the nucleotide specificity of synthesis enhancement, we used M13mp10 DNA containing 4 consecutive thymidine residues downstream from the 3-hydroxyl terminus of an oligonucleotide primer. Upon addition of factor D, both polymerase I and polymerase-alpha can traverse this sequence more efficiently and thus generate longer DNA products. Densitometric analysis of nonextended and elongated 5'-32P-labeled M13 primer indicates that, without changing the frequency of primer utilization, factor D enhances the activity of these DNA polymerases by increasing their apparent processivity. By positioning oligonucleotide primers 4, 8, and 12 bases upstream from the (dT)4 template sequence, we show that the enhancement of synthesis by factor D is independent of the position of the oligothymidine cluster. We hypothesize that factor D interacts with oligo(dT).oligo(dA) domains in DNA to alter their conformation, which may normally obstruct the progression of DNA polymerases.

Animals↗
PubMed1999

DNA sequence of the UL6 to UL20 genes of infectious laryngotracheitis virus and characterization of the UL10 gene product as a nonglycosylated and nonessential virion protein.

The 24 kbp KpnI restriction fragment A from the unique long genome region of infectious laryngotracheitis virus (ILTV, gallid herpesvirus-1) has been sequenced. The analysed region contains 14 open reading frames sharing homology with conserved alphaherpesvirus genes. Arrangement of the UL6 to UL20 homologues of ILTV is almost identical to that found in the herpes simplex virus type 1 genome. As in other herpesviruses the UL15 gene consists of two exons and is expressed from a spliced mRNA. However, the UL16 gene, which is usually localized within the intron sequence of UL15, is not conserved at this position of the ILTV genome. Another unique feature is the absence of any putative N-glycosylation motifs within the deduced ILTV UL10 gene product, which is the homologue of the conserved herpesvirus glycoprotein M. After preparation of a monospecific antiserum, two distinct UL10 proteins with apparent molecular masses of 36 and 31 kDa were identified in ILTV-infected cells as well as in purified virions. None of these UL10 gene products is modified by N- or O-linked glycosylation. Isolation of a green fluorescent protein-expressing UL10 deletion mutant of ILTV revealed that this gene is not required for virus replication in cell culture.

Animals↗
PubMed1989

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae.

The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT alpha, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.

Base Sequence↗
PubMed2000

Mitochondrial DNA sequence from an extinct population of European vendace, Coregonus albula L.

Nucleotide sequence of the mitochondrial DNA control region was determined for the formalin-preserved specimen of the individual European vendace, Coregonus albula, belonging to an extinct population. The specimen has been preserved and stored in formalin for 39 years. A comparison with two previously determined sequences obtained for specimens from other populations of vendace revealed both nucleotide substitutions and site heteroplasmy. The ability to sequence DNA from formalin-preserved museum specimens of rare or extinct taxa provides another sampling strategy for phylogenetic studies on fish.

Animals↗
PubMed1990

Chromosome ends in Drosophila without telomeric DNA sequences.

We have recovered terminal chromosome deletions of the X chromosome of Drosophila [Df(1)RT; RT = receding tips] that break in various positions of the yellow gene (y) region and delete all distal DNA sequences. Terminal DNA fragments are heterogeneous in length. Molecular cloning and sequencing of the terminal DNA fragments revealed that the broken ends of the deleted chromosomes do not carry any telomeric DNA sequences, yet the broken chromatids do not fuse to one another. Moreover, we confirmed by sequence analysis of 49 independently cloned terminal DNA fragments from two RT lines collected at different times that they lose DNA sequences from their distal ends at a rate of 70-75 base pairs per fly generation. We calculate that the rate of loss from these ends is consistent with the removal of an octanucleotide RNA primer at each round of DNA replication in the germ line.

Animals↗
PubMed1993

Simple, rapid, and accurate determination of deletion mutations by automated DNA sequencing of heteroduplex fragments of the adenomatous polyposis coli (APC) gene generated by PCR amplification.

Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were "bulge" heteroduplexes, while the other two were homoduplexes made up of two wild-type strands and two deletion-mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion-carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases.

Adenomatous Polyposis Coli↗
PubMed2001

DNA sequence copy number aberrations associated with histological subtypes and DNA ploidy in gastric carcinoma.

We have analyzed DNA sequence copy number aberrations (DSCNAs) and DNA ploidy by using comparative genomic hybridization and laser scanning cytometer in gastric carcinomas (GCs) to elucidate the genomic aberrations in relation to clinicopathological parameters. Thirty-two out of 33 cases showed one or more DSCNAs with a mean number of 11.7 per tumor. High-level gains were detected at 2p, 3q, 6p, 7p, 7q, 8q, 12p, 13q, 19q, and 20q. Frequency of gross genomic abnormalities and chromosome regions that have genomic aberrations were similar in both intestinal- and diffuse-type GCs, except aberrations at 8p, 9p, 12q, and 20q. The overall number of DSCNAs was significantly greater in DNA aneuploid tumors than that in DNA diploid tumors. We detected genomic aberrations characterized by histological subtype, tumor location, and DNA ploidy status: gain of 20q and losses of 8p and 9p in intestinal-type GCs, gains of 8p and 12q in diffuse-type GCs, gain of 20q in the lower third GCs, and loss of 5q, 9p, 10q, 16q, and 18q in DNA aneuploid GCs. Furthermore, 5q loss is associated with DNA aneuploidy (P = 0.0001) or the total number of losses (P = 0.001), gain + losses (P = 0.004), and high-level gains (P = 0.001) in GCs. Among these loci, chromosome 8p was unique. Gain of 8p was more common in diffuse-type GC, whereas loss of 8p was more frequently detected in intestinal-type GC. In conclusion, we describe chromosomal regions of 5q, 8p, and 20q, which are of interest for further investigation of GCs.

Adenocarcinoma↗
PubMed2003

Human inter-individual DNA sequence variation in candidate genes, drug targets, the importance of haplotypes and pharmacogenomics.

The identification of genes predisposing to human diseases is of paramount importance for understanding the molecular basis of the disease and individually different drug response, and will establish new routes to diagnosis and therapeutic advances of immense medical benefit. A key step common to all strategies for disease gene identification is the systematic analysis of candidate gene sequences to identify specific sequence variations associated with disease or any other phenotype of pharmaceutical relevance. In this article, current concepts and approaches to haplotype-based candidate gene analysis are reviewed. Moreover, a comprehensive summary of recent studies and data on the amount, nature, pattern and structure of genetic variation in candidate genes is given. These data demonstrate altogether remarkable gene sequence and haplotype diversity. Numerous individually different forms of a gene may exist. This presents challenges to the traditional views of the concept of "a" gene with far-reaching implications on the functional analysis of candidate gene variation, on the establishment of "sequence"-"structure"-"function" and complex haplotype/genotype-phenotype relationships, on the identification, evaluation and prioritization of drug targets and the concept of a "personalized medicine" in general. Moreover, present and future approaches to the identification of candidate and disease genes will be addressed. These include whole genome-based approaches such as integrative genomics as well as functional genomics-based approaches to analyze and model complex biological and medical processes. The analysis of whole complex systems in particular will provide the basis to make "maximally informed" guesses on candidate genes and address complex variability patterns in genes as well as complex genotype-phenotype relationships comprehensively at an advanced level.

Base Sequence↗
PubMed1996

Catalytic activity of poly(ADP-ribose) polymerase is necessary for repair of N-methylpurines in nontranscribed, but not in transcribed, nuclear DNA sequences.

The role of poly(ADP-ribose) polymerase (PADPRP) in nuclear DNA repair and other nuclear processes has been intensely studied and debated for decades. Recent studies have begun to shed new light on these arguments with firm experimental data for its role, primarily, as a remodeler of chromatin structure. Those studies imply that PADPRP plays an indirect role in DNA repair, serving to expose DNA to repair enzymes through chromatin remodeling. Only DNA that is tightly packaged would require PADPRP activity for its repair; while DNA in an open conformation would be accessible to DNA repair enzymes and not require PADPRP activity. The purpose of the current studies was to address the above hypothesis directly. Using quantitative Southern blot analysis, we studied repair in transcribed and nontranscribed nuclear DNA sequences in ADPRT 351 cells 95% deficient in PADPRP activity. Cells were exposed to methylnitrosourea (MNU) for 1 h and allowed to repair for 8 or 24 h. Densitometric scans of autoradiographs revealed that, when compared to their parental V79 cell line, ADPRT 351 cells 95% deficient in PADPRP activity were equally as efficient in repair of N-methylpurines in the transcribed sequence containing the dihydrofolate reductase gene. However, the ADPRT 351 cells were deficient in the ability to repair these lesions in the nontranscribed sequence containing the IgE gene compared to repair of the same sequence in the parental V79 cells. Nucleoid sedimentation assays demonstrated that the ADPRT 351 cells are deficient in repair across the entire genome when compared to the parental V79 cells. These studies indicate that PADPRP activity is not required for repair of N-methylpurines in transcribed nuclear DNA sequences but is necessary for the repair of these lesions in nontranscribed nuclear DNA sequences as well as across the entire genome since the DNA in a given cell is predominantly nontranscribed.

Alkylation↗
PubMed1982

A DNA sequence handling program.

A computer program that aids in recording, editing, and analysis of the base sequences of DNA and RNA is presented. A tape containing copies of the program and the user manual for it are available at cost.

Base Sequence↗
PubMed2003

Primer design for automated DNA sequencing in a core facility.

We assessed the quality of nine standard primers for automated fluorescent dye terminator DNA sequencing by whether their melting temperatures (Tms) were in the optimal range for DNA sequencing, and the degree to which their sequences matched the sequences of 36 common vectors. The M13F (-21/-20), M13F (-41/-40), M13R, and T7R primers showed optimal physicochemical characteristics and were not redesigned. The M13R (-41/-40), T3, and SP6 primers showed mismatches and/or Tm values outside of the optimal range and were redesigned by these two criteria. With few exceptions, the redesigned primers did not significantly improve DNA sequencing quality as assessed by Phred scores compared to the corresponding original primer. However, both redesigned T7 primers, which were also redesigned to function as pET primers, strikingly improved sequencing on pET vectors. The original and redesigned T7 primers also improved the sequence quality for the Bluescript family of vectors compared to the original and redesigned M13R (-41/-40) primers. The mismatch-redesigned T7, SP6, and M13R (-41/-40) primers marginally improved the sequence quality over the corresponding original primers for specific vector types. Within limits, decreasing the %GC and Tm below reported optimal levels did not affect the sequence quality. Commercially available standard vector primers were surveyed.

Base Sequence↗
PubMed1977

DNA sequencing with chain-terminating inhibitors.

A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.

Base Sequence↗
PubMed1994

DNA sequence confidence estimation.

A significant bottleneck in the current DNA sequencing process is the manual editing of trace data generated by automated DNA sequencers. This step is used to correct base calls and to associate to each base call a confidence level. The confidence levels are used in the assembly process to determine overlaps and to resolve discrepancies in determining the consensus sequence. This single step may cost as much as 4 to 8 cents per finished base. We report an approach to automated trace editing using classification trees to detect and exploit context-based patterns in trace peak heights. Local base composition and nearby peak heights account for 80% of the variations in peak heights. Classification algorithms were developed to identify 37% of automated base calls that differ from the consensus sequence. With these algorithms, 12% of the base calls had confidence levels less than 90%.

Algorithms↗
PubMed2002

TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing.

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.

Bacillus Phages↗
PubMed1991

Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis.

Multiwavelength detection of laser induced fluorescence for dideoxynucleotide DNA sequencing with four different fluorophores and separation by capillary gel electrophoresis is described. A cryogenically cooled, low readout noise, 2-dimensional charge-coupled device is used as a detector for the on-line, on-column recording of emission spectra. The detection system has no moving parts and provides wavelength selectivity on a single detector device. The detection limit of fluorescently labeled oligonucleotides meets the high sensitivity requirements for capillary DNA sequencing largely due to the efficient operation of the CCD detector with a 94% duty cycle. Using the condition number as a selectivity criterion, multiwavelength detection provides better analytical selectivity than detection with four bandpass filters. Monte Carlo studies and analytical estimates show that base assignment errors are reduced with peak identification based on entire emission spectra. High-speed separation of sequencing samples and the treatment of the 2-dimensional electropherogram data is presented. Comparing the DNA sequence of a sample separated by slab gel electrophoresis with sequence from capillary gel electrophoresis and multiwavelength detection we find no significant difference in the amount of error attributable to the instrumentation.

Base Sequence↗
PubMed1971

Hybridization properties of DNA sequences directing the synthesis of messenger RNA and heterogeneous nuclear RNA.

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of D(o)t (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of D(o)t about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11 degrees C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4 degrees -6 degrees C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA.

Animals↗