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At least 19 recordsLinked to original sources
PubMed1996

Repetitive and arbitrary primer DNA sequences in PCR-mediated fingerprinting of outbreak and sporadic isolates of Campylobacter jejuni.

PCR-mediated fingerprinting with combined repetitive and arbitrary DNA primers (ERIC-2 and 1026) was used to type Campylobacter jejuni from a milk-associated outbreak, and from sporadic cases of the same and allied HS serotypes. The 14 outbreak strains had identical or similar DNA band profiles whereas the 25 strains from sporadic infections were more heterogeneous with 18 different DNA profiles. Although PCR-based DNA fingerprints lacked serotype specificity, the method was fast, simple to perform and reproducible, provided defined technical protocols were adhered to precisely. Profiles were highly discriminatory but did not consistently match types based on other molecular methods. We conclude that AP-PCR has demonstrable potential for initial rapid investigation of outbreaks, and when used in conjunction with PFGE analysis of DNA restriction profiles, provides a high resolution strategy for accurately defining subtypes of C. jejuni.

Animals↗
PubMed2005

Universal DNA primers amplify bacterial DNA from human fetal membranes and link Fusobacterium nucleatum with prolonged preterm membrane rupture.

A large number of bacterial species have been identified in fetal membranes after preterm labour (PTL) associated with intrauterine infection by microbiological culture. In this study, we have investigated a molecular and bioinformatic approach to organism identification which surmounts the need for specific and diverse microbiological culture conditions required by conventional methods. Samples of fetal membranes were taken from 37 preterm infants, and 6 normal term controls delivered by caesarean section, in which bacteria had been detected by in situ hybridization of 16S ribosomal RNA using a generic probe. Degenerate primers were designed to amplify bacterial 16S ribosomal DNA by PCR and used to amplify bacterial DNA from human fetal membranes. Amplicons were cloned, sequenced and bacteria were identified bioinformatically by comparison of sequences with known bacterial DNA genomes. In situ hybridization using an organism specific probe was then used to confirm the presence of the commonest identified organism in tissue samples. Bacterial DNA amplified from 15/43 samples, all from preterm deliveries, and the bioinformatic approach identified organisms in all cases. Multiple bacteria were identified including Mycoplasma hominis, Pasturella multocida, Pseudomonas PH1, Escherichia coli and Prevotella bivia. The commonest organism Fusobacterium nucleatum was found in 9/15 (60%) of samples. Ten of the 12 samples obtained after prolonged membrane rupture were positive for bacterial DNA, and 7 of these (70%) contained DNA from F. nucleatum. Bacteria from fetal membranes may be identified by molecular and bioinformatic methods. Further work is warranted to investigate the apparent linkage between F. nucleatum, fetal membrane rupture and preterm delivery.

Bacterial Typing Techniques↗
PubMed1993

Eliminating mitochondrial DNA competition for nuclear DNA primers.

Mitochondrial DNA (mtDNA) sequences were synthesized with nuclear DNA (nucDNA) sequence-tagged site (STS) primers by mismatch priming in three independent studies of the human nuclear genome. Mismatch primer binding sites on the mtDNA were identified with from 6- to 10-bp identity at the 3' ends of the primers. In two of three cases, single-stranded mtDNA copies were gel-isolated with intended nucDNA PCR products. During routine screening of the STSs, the radiolabeled gel-isolated products hybridized to polymorphic mtDNA restriction fragments. Intense signals after overnight exposure of radiolabeled PCR probes on Southern blots suggest contaminating mtDNA PCR products. The theoretical annealing temperatures of the mismatches were well below the annealing temperatures of the PCR primers, demonstrating annealing reactions driven by the molar surplus of the primers, that is, mass action. The probability that two primers (either one of a pair or both), designed to amplify nucDNA, will bind to and amplify mtDNA may be as high as 1 in 64, assuming that an identical match with only the 3' hexanucleotide is sufficient for amplification. To circumvent this problem we have developed OLIGFIND, a program that has identified the 104 of 4096 possible hexamers that are not present in human mtDNA. Our results suggest that time could be saved by designing STS primers with one of these 104 hexamers at the 3' end. OLIGFIND can also evaluate primer 3' ends for potential PCR products from mtDNA.

Base Sequence↗
PubMed1985

The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis.

A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety of electrophiles, thereby allowing other chemical species to be attached to the oligonucleotide. This chemistry has been utilized to synthesize several fluorescent derivatives of an oligonucleotide primer used in DNA sequence analysis by the dideoxy (enzymatic) method. The modified primers are highly fluorescent and retain their ability to specifically prime DNA synthesis. The use of these fluorescent primers in DNA sequence analysis will enable DNA sequence analysis to be automated.

Base Sequence↗
PubMed1993

Primer-DNA formation during simian virus 40 DNA replication in vitro.

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.

Antigens, Neoplasm↗
PubMed1992

Assembly of simian virus 40 Okazaki pieces from DNA primers is reversibly arrested by ATP depletion.

We have previously proposed that DNA polymerase alpha-primase provides short RNA-DNA precursors below 40 nucleotides (DNA primers), several of which assemble into an Okazaki piece after intervening RNA has been removed and the gaps have been filled by DNA polymerase delta (or epsilon) (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988; T. Nethanel and G. Kaufmann, J. Virol. 64:5912-5918, 1990). In this report, we confirm and extend these conclusions by studying the effects of deoxynucleoside triphosphate (dNTP) concentrations and the presence of ATP on the occurrence, dynamics, and configuration of DNA primers in simian virus 40 replicative intermediate DNA. We first show that these parameters are not significantly affected by a 10-fold increase in dNTP precursor concentrations. We then demonstrate that Okazaki piece synthesis can be arrested at the level of DNA primers by ATP depletion. The arrested DNA primers faced short gaps of 10 to 20 nucleotides at their 3' ends and were progressively chased into Okazaki pieces when ATP was restored. ATP could not be substituted in this process by adenosine-5'-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. The chase was interrupted by aphidicolin but not by butylphenyl-dGTP. The results implicate an ATP-requiring factor in the switch between the two DNA polymerases engaged in Okazaki piece synthesis. They also suggest that the replication fork advances by small, DNA primer-size increments.

Adenosine Triphosphate↗
PubMed1999

Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein. Template-primer DNA binding and initiation and elongation of DNA strand synthesis.

Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos have been evaluated with regard to the overall activity of pol gamma and in partial reactions involving template-primer binding and initiation and idling in DNA strand synthesis. Both the 5' --> 3' DNA polymerase and 3' --> 5' exonuclease in pol gamma are stimulated 15-20-fold on oligonucleotide-primed single-stranded DNA by native and recombinant forms of mtSSB. That the extent of stimulation is similar for both enzyme activities over a broad range of KCl concentrations suggests their functional coordination and a similar mechanism of stimulation by mtSSB. At the same time, the high mispair specificity of pol gamma in exonucleolytic hydrolysis is maintained, indicating that enhancement of pol gamma catalytic efficiency is likely not accompanied by increased nucleotide turnover. DNase I footprinting of pol gamma.DNA complexes and initial rate measurements show that mtSSB enhances primer recognition and binding and stimulates 30-fold the rate of initiation of DNA strands. Dissociation studies show that productive complexes of the native pol gamma heterodimer with template-primer DNA are formed and remain stable in the absence of replication accessory proteins.

Animals↗
PubMed2000

Specific detection of Leuconostoc mesenteroides subsp. mesenteroides with DNA primers identified by randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA analysis using primer 239 (5' CTGAAGCGGA 3') was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.

DNA Primers↗
PubMedDate not supplied

Development of mitochondrial DNA primers for use with homarid lobsters.

DNA primer sets were developed for the amplification of complete mitochondrial genomes for both European and American lobsters in 4 suitable-sized segments. Optimal conditions for polymerase chain reaction routine screening were established. The 4 segments were screened with 24 restriction endonucleases in a test population sample, covering the whole distribution of the European lobster, and restriction patterns of each enzyme were revealed. A segment of 3000 bp comprising part of cytochrome oxidase I gene, the genes cytochrome oxidase II and III, subunits 6 and 8 of ATPase, subunit 3 of the NAD dehydrogenase, and various transfer RNAs, was found to be the most polymorphic. A number of enzyme patterns in each segment differentiated European and American lobsters. Extra bands were observed, indicating heteroplasmy phenomena, which were verified with various approaches. Furthermore, a primer set that enables 1-step ampli fication of the complete mitochondrial genome of the European lobster was established.

Animals↗
PubMed1998

Negative regulation of yeast telomerase activity through an interaction with an upstream region of the DNA primer.

A number of published studies indicate that telomerase may interact with oligonucleotide primers in a bipartite manner, with the 3'-end of the primer positioned at the catalytic site of the enzyme and a more 5' region of the primer binding to a second or 'anchor' site of the enzyme. We systematically investigated the effects of mutations in the DNA primer on overall binding and polymerization by yeast telomerase. Our studies indicate that there is sequence-specific interaction between telomerase and a substantial region of the DNA primer. Mutations in the 3'-most positions of the primer reduced polymerization, yet had little effect on overall binding affinity. In contrast, mutations around the -20 position reduced binding affinity but had no effect on polymerization. Most strikingly, mutations centered around the -12 position of the DNA primer reduced overall binding affinity but dramatically enhanced primer extension, as well as primer cleavage. This finding suggests that reduced interaction with the -12 region of the DNA primer can facilitate a step in the catalytic region of yeast telomerase that leads to greater polymerization. A tripartite model of interaction between primer and telomerase is proposed to account for the distinct effects of mutations in different regions of the DNA primer.

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

Sensitive and specific detection of Xanthomonas campestris pv. pelargonii with DNA primers and probes identified by random amplified polymorphic DNA analysis.

The random amplified polymorphic DNA method was used to distinguish strains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomonas species and/or pathovars. Among the 42 arbitrarily chosen primers evaluated, 3 were found to reveal diagnostic polymorphisms when purified DNAs from compared strains were amplified by the PCR. The three primers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from various locations worldwide. The distinctive X. compestris pv. pelargonii patterns were clearly different from those obtained with any of 46 other Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, apparently unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA probe. It hybridized with total DNA from all 53 X. campestris pv. pelargonii strains tested and not with any of the 46 other Xanthomonas strains tested. The DNA sequence of the terminal ends of this 1.2-kb fragment was obtained and used to design a pair of 18-mer oligonucleotide primers specific for X. campestris pv. pelargonii. The custom-synthesized primers amplified the same 1.2-kb DNA fragment from all 53 X. campestris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophytes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the custom-synthesized primers was between 10 and 50 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMedDate not supplied

Localization of the large subunit of replication factor C near the 5' end of DNA primers.

Replication factor C (RFC) is a heteropentameric sliding clamp loader protein essential for processive synthesis of DNA by eukaryotic DNA polymerases delta and epsilon. To study the interaction of RFC with 3' and 5' ends of the DNA primer, we have developed chemical photocrosslinking assay using a synthetic DNA gap and DNA primer-template structures. We have found that the radioactively labeled primers containing a photoreactive group at their 5' end could crosslink with the largest RFC subunit (RFC140) on primer-templates and DNA gap structures, but that 3' end photoreactive primers could only crosslink with RFC140 within the DNA gap structure. Addition of replication protein A (RPA) to the reaction mixture resulted in the crosslinking of RPA subunits and inhibited crosslinking of RFC140 using 3' but not 5' photoreactive primers present at the gap. The results suggest specific contacts between RFC140 and the 5' end of the DNA primer. Together with previous data, these experiments allow us to propose a model for the DNA polymerase switch during eukaryotic DNA replication.

5' Flanking Region↗