Showing papers on "genomic DNA published in 1999"

Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes.

Abstract: Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

Fluorescence Polarization in Homogeneous Nucleic Acid Analysis

Abstract: A new method for DNA diagnostics based on template-directed primer extension and detection by fluorescence polarization is described. In this method, amplified genomic DNA containing a polymorphic locus is incubated with oligonucleotide primers (designed to hybridize to the DNA template adjacent to the polymorphic site) in the presence of allele-specific dye-labeled dideoxyribonucleoside triphosphates and a commercially available modified Taq DNA polymerase. The primer is extended by the dye-terminator specific for the allele present on the template, increasing approximately 10-fold the molecular weight of the fluorophore. At the end of the reaction, the fluorescence polarization of the two dye-terminators in the reaction mixture are analyzed directly without separation or purification. This homogeneous DNA diagnostic method is shown to be highly sensitive and specific and is suitable for automated genotyping of large number of samples. [The data shown in Figure 3 are available as an online supplement at.]

Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87

Abstract: The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, and phlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.

Quantitation of Hepatitis B Virus Genomic DNA by Real-Time Detection PCR

Abstract: Quantitation of hepatitis B virus (HBV) DNA in serum is a useful method for the monitoring of HBV replication. We attempted to develop a quantitative assay system for HBV DNA that is more sensitive, accurate, and reproducible than existing systems. We detected HBV DNA by real-time detection PCR (RTD-PCR) based on Taq Man chemistry. The efficacy of this assay was evaluated by quantitatively measuring sequential levels of synthetic DNA and DNA in clinical serum samples. The detection limit of this system was as few as 10 DNA copies/reaction. A linear standard curve was obtained between 101 and 108 DNA copies/reaction. The coefficient of variation for both intra- and interexperimental variability indicated remarkable reproducibility. This system detected HBV DNA in 100% of chronic hepatitis B patients tested and never detected HBV DNA in healthy volunteers who were negative for HBV markers. These observations suggest that RTD-PCR is an excellent candidate for a standard HBV quantification method.

Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning.

Abstract: To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable. Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. Furthermore, it has the cis sequences required for Agrobacterium-mediated gene transfer into plants. We cloned large genomic DNA fragments of Arabidopsis thaliana into the vector and showed that most of the DNA fragments were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we demonstrated the practical utility of this vector system for positional cloning in Arabidopsis. A TAC contig was constructed in the region of the SGR1 locus, and individual clones with ca. 80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line. Successful complementation enabled the physical location of SGR1 to be delimited with high precision and confidence.

Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.

Abstract: A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in Escherichia coli. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions. The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection. Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye. Mutagenesis of pBecker1 in E. coli with a mini-Tn5-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants. The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable. However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells. The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed.

Molecular Typing of S-alleles through Identification, Characterization and cDNA Cloning for S-RNases in Sweet Cherry

Abstract: This report identifies S-RNases of sweet cherry (Prunus avium L.) and presents information about cDNA sequences encoding the S-RNases, which leads to the development of a molecular typing system for S-alleles in this fruit tree species. Stylar proteins of sweet cherry were surveyed by two dimensional polyaclylamide gel electrophoresis (2D- PAGE) to identify S-proteins associated with gametophytic self-incompatibility. Glycoprotein spots linked to S-alleles were found in a group of proteins which had Mr and pI similar to those of other rosaceous S-RNases. These glycoproteins were present at highest concentration in the upper segment of the mature style and shared immunological characteristics and N-terminal sequences with those of S-RNases of other plant species. cDNAs encoding these glycoproteins were cloned based on the N-terminal sequences. Genomic DNA and RNA blot analyses and deduced amino acid sequences indicated that the cDNAs encode S-RNases; thus the S-proteins identified by 2D-PAGE are S-RNases. Although S 1 to S 6 -alleles of sweet cherry cultivars could be distinguished from each other with the genomic DNA blot analysis, a much simpler method of PCR-based typing system was developed for the six S-alleles based on the DNA sequence data obtained from the cDNAs encoding S-RNases.

Discovery and Characterization of Polymorphic Simple Sequence Repeats (SSRs) in Peanut

Abstract: Cultivated peanut (Arachis hypogaea L), an important agronomic crop, exhibits a considerable amount of variability for morphological traits and for resistance to diseases and pests. In contrast, molecular marker assays have detected little variation at the nucleic add level. Identification of molecular markers would be of great help to peanut breeders, geneticists, and taxonomists. The objectives of this work were to identify simple sequence repeat (SSR) markers in cultivated peanut and to test these markers for their ability to discriminate among accessions. Peanut total genomic DNA libraries were constructed and screened with 32 P-labeled dinucleotide repeats, (GT) 10 and (CT) 10 . DNA sequences were obtained from the SSR-containing clones and, when possible, primer pairs were designed on the basis of DNA sequences flanking the repeat motif. Primer pairs were tested in polymerase chain reaction (PCR) assays using a collection of 22 peanut DNAs, representing both cultivated peanut and wild species. In all, six SSR markers, five from the library screening procedure and one additional marker obtained from a search of publicly available DNA sequences, detected polymorphisms among the peanut DNAs. Discrimination power was high among the cultivated peanuts, with 17 unique genotypes represented among the 19 accessions tested. From two to 14 DNA fragments were amplified per SSR marker, and as a group, the six markers may amplify up to 10 putive SSR loci. The SSR markers identified in this study were more effective in detecting molecular variation in cultivated peanut than all other DNA-based markers evaluated to date.

Highly Sensitive and Specific Fluorescence Reverse Transcription-PCR Assay for the Pseudogene-free Detection of β-Actin Transcripts as Quantitative Reference

Abstract: The use of common reverse transcription (RT)-PCR reference target sequences can produce false-positive results by amplification of either contaminating DNA or processed pseudogenes. Furthermore, qualitative RT-PCR alone cannot distinguish between high- and poor-quality cDNA preparations, which again may be crucial for the interpretation of low-abundance transcripts. We have developed a highly sensitive quantitative RT-PCR for β-actin, using the TaqManTM chemistry. Through this technique, we were able to quantitatively detect 10 β-actin molecules per 100 ng of cDNA without coamplification of pseudogenes or genomic DNA. Thus, the presented method may be advantageous for the interpretation of quantitative RT-PCR results. PCR analysis may be difficult to interpret unless a reference target sequence is amplified in parallel. This control reaction is necessary to evaluate whether a sufficient amount of amplifiable material is present in the sample investigated. Thus, negative PCR can be defined as such only when amplification of a reference gene reveals a positive result. Amplifying a reference is especially important in RT-PCR because RNA can be degraded rapidly before or during cDNA synthesis. As reference sequences for RT-PCR, so called “housekeeping” genes are preferred because they are constitutionally expressed by all cell types. β-Actin is an attractive candidate for reference coamplification because it exhibits only minor intraindividual kinetic changes and is not primarily affected by any human disease (1). However, a major concern with β-actin and other commonly used references such as glyceraldehyde-3-phosphate dehydrogenase is that processed pseudogenes can be coamplified by primers that are originally restricted to cDNA. This problem is difficult to circumvent because sequence homologies of >90% exist between mRNA and the respective pseudogenes (2); thus, the pseudogenes usually cannot be distinguished in gel electrophoresis. Furthermore, as long as genomic DNA contamination cannot safely be excluded, DNA may be coamplified together with cDNA. Thus, positive analysis of …

Whole-genome shotgun optical mapping of Deinococcus radiodurans.

Abstract: A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after γ irradiation.

Correction of the UDP-glucuronosyltransferase gene defect in the gunn rat model of crigler-najjar syndrome type I with a chimeric oligonucleotide.

Abstract: Crigler–Najjar syndrome type I is characterized by unconjugated hyperbilirubinemia resulting from an autosomal recessive inherited deficiency of hepatic UDP-glucuronosyltransferase (UGT) 1A1 activity. The enzyme is essential for glucuronidation and biliary excretion of bilirubin, and its absence can be fatal. The Gunn rat is an excellent animal model of this disease, exhibiting a single guanosine (G) base deletion within the UGT1A1 gene. The defect results in a frameshift and a premature stop codon, absence of enzyme activity, and hyperbilirubinemia. Here, we show permanent correction of the UGT1A1 genetic defect in Gunn rat liver with site-specific replacement of the absent G residue at nucleotide 1206 by using an RNA/DNA oligonucleotide designed to promote endogenous repair of genomic DNA. The chimeric oligonucleotide was either complexed with polyethylenimine or encapsulated in anionic liposomes, administered i.v., and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, and DNA sequencing, and confirmed by genomic Southern blot analysis. DNA repair was specific, efficient, stable throughout the 6-month observation period, and associated with reduction of serum bilirubin levels. Our results indicate that correction of the UGT1A1 genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, with consequent improvement in the metabolic abnormality.

Genotyping by mass spectrometric analysis of short dna fragments

Abstract: Genotyping can be accomplished by analysis of short, defined DNA segments using electrospray ionization mass spectrometry. The DNA segments are produced using specially designed primers to amplify a cDNA or genomic DNA template. The primers contain a recognition site for a restriction endonuclease. The amplification products are digested with the restriction endonuclease. Single nucleotide polymorphisms can be detected rapidly and reliably.

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

Abstract: The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT) poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC)poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures

Plasmid DNA Malaria Vaccine: The Potential for Genomic Integration after Intramuscular Injection

Abstract: Plasmid-based (naked DNA) genetic vaccines are now entering clinical trials to test their safety and efficacy in healthy human volunteers. A safety concern unique to this new class of vaccines is the potential risk of deleterious integration into host cell genomic DNA following direct intramuscular injection. To address this issue experimentally, a preclinical safety study was conducted in mice to determine the structural nature of plasmid DNA sequences persisting in total muscle DNA at both 30 and 60 days following a single intramuscular injection of a plasmid expressing the Plasmodium falciparum circumsporozoite protein. In a protocol described for the first time, total DNA was extracted from muscle tissue and was subsequently linearized with a restriction endonuclease to enable agarose gel size fractionation of all extrachromosomal plasmid DNAs from high molecular weight mouse genomic DNA. Using PCR assays to quantitate plasmid-specific sequences, it was found that the amount of plasmid DNA persisting in muscle tissue varied but averaged about 10 fg per microgram of genomic DNA (in the range of 1500 copies per 150,000 genomes). In two of four separate experimental injections of mouse muscle, PCR assays of genomic DNA fractions indicated that agarose gel purification removed plasmid DNA down to a level of < or =3 copies per 150,000 mouse genomes. In the two other experimental samples, 3-30 copies of plasmid DNA remained associated with purified genomic DNA. The time following injection (i.e., 30 or 60 days) was not a factor in the number of copies of plasmid associating with genomic DNA and it was not possible to conclude if such sequences were covalently linked to genomic DNA or simply adventitiously associated with the genomic DNA. However, if an assumption is made that the highest level plasmid DNA found associated with genomic DNA (i.e., 30 copies) represented covalently integrated plasmid inserts and that each insert resulted in a mutational event, the calculated rate of mutation would be 3000 times less than the spontaneous mutation rate for mammalian genomes. This level of integration, if it should occur, was not considered to pose a significant safety concern.

Sequence-tagged connectors: A sequence approach to mapping and scanning the human genome

Abstract: The sequence-tagged connector (STC) strategy proposes to generate sequence tags densely scattered (every 3.3 kilobases) across the human genome by arraying 450,000 bacterial artificial chromosomes (BACs) with randomly cleaved inserts, sequencing both ends of each, and preparing a restriction enzyme fingerprint of each. The STC resource, containing end sequences, fingerprints, and arrayed BACs, creates a map where the interrelationships of the individual BAC clones are resolved through their STCs as overlapping BAC clones are sequenced. Once a seed or initiation BAC clone is sequenced, the minimum overlapping 5′ and 3′ BAC clones can be identified computationally and sequenced. By reiterating this “sequence-then-map by computer analysis against the STC database” strategy, a minimum tiling path of clones can be sequenced at a rate that is primarily limited by the sequencing throughput of individual genome centers. As of February 1999, we had deposited, together with The Institute for Genomic Research (TIGR), into GenBank 314,000 STCs (≈135 megabases), or 4.5% of human genomic DNA. This genome survey reveals numerous genes, genome-wide repeats, simple sequence repeats (potential genetic markers), and CpG islands (potential gene initiation sites). It also illustrates the power of the STC strategy for creating minimum tiling paths of BAC clones for large-scale genomic sequencing. Because the STC resource permits the easy integration of genetic, physical, gene, and sequence maps for chromosomes, it will be a powerful tool for the initial analysis of the human genome and other complex genomes.

Complexity management and analysis of genomic DNA

Abstract: The present invention provides for novel methods of sample preparation and analysis involving reproducibly reducing the complexity of a nucleic sample. The invention further provides for analysis of the above sample by hybridization to an array which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the presence or absence of a polymorphism. The invention further provides for novel methods of using a computer system to model enzymatic reactions in order to determine experimental conditions before conducting actual experiments.

Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT7 intron and exon sequences

Abstract: The phylogenetic position of Candida dubliniensis has previously been established on the basis of the sequence of rRNA genes. In order to confirm the relationship between C. dubhiensis and other yeast species, particularly Candida a/bicans, using non-rRNA gene sequences the ACT1 gene was chosen for analysis. Three overlapping fragments that together span the entire C. dubhiensis ACT1 gene (CdACT1) were amplified from a recombinant phage isolated from a genomic DNA A library using PCR. These were cloned and used to determine the contiguous sequence of the gene. Analysis of the sequence data revealed the presence of a 1131 bp ORF interrupted by a single 632 bp intron at the 5' extremity of the gene. Comparison of the CdACT1 sequence with the C. albicans homologue (CaACT1) revealed that although the exons are 97-9 O/O identical the introns are only 83.4% identical. Phylogenetic trees generated using ACT1 exon and intron sequences from a range of yeast species unequivocally confirmed the phylogenetic position of C. dubhiensis as a unique taxon within the genus Candida. Analysis of the ACT1-associated intron sequences from 10 epidemiologically unrelated C. dubhiensis isolates from disparate geographical locations showed a very low level of intraspecies sequence variation. In order to develop an accurate and rapid method to identify C. dubliniensis from primary isolation plates the significant divergence between the C. dubhiensis and C. albicans ACT1 intron sequences was exploited by designing C. dubliniensis-specific PCR primers. Using a rapid boiling method to produce template DNA directly from colonies from primary isolation plates in 10 min, these primers were used in a blind test with 122 isolates of C. dubhiensis, 53 isolates of C. albicans, 10 isolates of C. stellatoidea and representative isolates of other clinically relevant Candida and other yeast species. Only the C. dubhiensis isolates yielded the C. dubhiensis-specif ic 288 bp amplimer. Use of this technique on colonies suspected to be C. dubhiensis allows their correct identification as C. dubhiensis in as little as 4 h.

Nucleic acid analysis using sequence-targeted tandem hybridization

Abstract: The disclosed invention provides a novel method for analyzing genomic DNA and expressed sequences using auxiliary oligonucleotides, preannealed to the single-stranded target nucleic acid to form a partially duplex target molecule, offers several advantages in the analysis of nucleic acid sequences by hybridization to genosensor arrays or “DNA chips”. Also provided is a method for directly analyzing and comparing patterns of gene expression at the level of transcription in different cellular samples.

Detection of aneuploidy in single cells using comparative genomic hybridization.

Abstract: The ability of comparative genomic hybridization (CGH) to detect aneuploidy following universal amplification of DNA from a single cell, or a small number of cells, was investigated with a view to preimplantation diagnosis following in vitro fertilization, and prenatal diagnosis using fetal erythroblasts obtained from maternal blood. The DNA obtained from lysed single cells was amplified using degenerate oligonucleotide-primed PCR (DOP-PCR). This product was labelled using nick translation and hybridized together with normal reference genomic DNA. The CGH fluorescent ratio profiles obtained could be used to determine aneuploidy with cut-off thresholds of 0.75 and 1.25. Deviation in the profiles in the heterochromatic regions was reduced by using, as a reference sample, normal genomic DNA that had also undergone DOP-PCR. Single cells known to be trisomic for chromosomes 13, 18 or 21 were analysed using this technique. The resolution of CGH with amplified DNA from a single cell is of the order of 40 Mb, sufficient for the diagnosis of trisomy 21, and possibly segmental aneuploidy of equivalent size. These results, and those of others, demonstrate that diagnosis of chromosomal aneuploidy in single cells is possible using CGH with DOP-PCR amplified DNA.