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At least 73 records · Page 4Linked to original sources
PubMed1997

Classification of plant trypanosomatids (Phytomonas spp.): parity between random-primer DNA typing and multilocus enzyme electrophoresis.

The genetic polymorphism of 30 isolates of plant trypanosomatids colloquially referred to as plant trypanosomes was assayed by means of RAPD. The principle objectives of this study were to assess the discriminative power of RAPD analysis for studying plant trypanosomes and to determine whether the results obtained were comparable with those from a previous isoenzyme (MLEE) study. The principle groups of plant trypanosomes identified previously by isoenzyme analysis--intraphloemic trypanosomes, intralaticiferous trypanosomes and trypanosomes isolated from fruits--were also clearly separated by the RAPD technique. Moreover, the results showed a fair parity between MLEE and RAPD data (coefficient of correlation = 0.84) and the two techniques have comparable discriminative ability. Most of the separation revealed by the two techniques between the clusters was associated with major biological properties. However, the RAPD technique gave a more coherent separation than MLEE because the intraphloemic isolates, which were biologically similar in terms of their specific localization in the sieve tubes of the plant, were found to be in closer groups by the RAPD. For both techniques, the existence of the main clusters was correlated with the existence of synapomorphic characters, which could be used as powerful tools in taxonomy and epidemiology.

Animals↗
PubMed1995

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Bacteriophage M13↗
PubMed1997

Ligation mediated fluorescent labeling of DNA sequencing primers.

Automated DNA sequencing utilizing fluorescently labeled primers is a proven methodology for generating quality sequence data. However, for directed primer walking strategies this necessitates synthesis and labeling of a unique primer for each sequencing reaction. Here, we describe a rapid ligation-based method of generating labeled sequencing primers. An unlabeled 5'-phosphorylated sequencing primer is ligated to a fluorescent oligonucleotide by use of a bridge primer which is complementary to portions of the previous two oligonucleotides, thus aligning them properly for ligation. The resulting fluorescent hybrid primer can be utilized directly in cycle sequencing reactions without any prior purification.

Chromosome Walking↗
PubMed1978

DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities.

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.

Chlamydomonas↗
PubMedDate not supplied

DNA amplification fingerprinting using arbitrary oligonucleotide primers.

DNA amplification fingerprinting (DAF) is a strategy for genetic typing and mapping that uses one or more very short (> or = 5 nt) arbitrary oligonucleotides to direct the enzymatic amplification of discrete portions of a DNA template resulting in a spectrum of products characteristic of the DNA starting material. Polymorphisms from simple banding patterns are useful as genetic markers while more complex and informative patterns are suitable for DNA fingerprinting. The use of polyacrylamide gel electrophoresis and silver staining can adequately resolve the spectrum of DAF products into detailed and reproducible patterns.

Base Sequence↗
PubMed2006

Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer.

DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO PrimeSurface with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3' end of the immobilized DNA primers on the S-Bio by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.

DNA Primers↗
PubMed2006

Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation.

The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The attachment of long single-strands on GCE surface caused the accumulation of [Co(NH3)6]3+ and a high current response. Here, we report a versatile method that would allow for simple and rapid analysis of nucleic acids in combination with PNA-mediated PCR and asymmetric PCR techniques by using an electrochemical genosensor.

Base Sequence↗
PubMed2003

Comparison of polymerase chain reaction methods for reliable and easy detection of congenital Trypanosoma cruzi infection.

The polymerase chain reaction (PCR) is a potentially interesting diagnostic tool for detecting congenital Trypanosoma cruzi infection at birth. We have compared the sensitivity and capacity of a group of T. cruzi PCR primers in detecting the complete spectrum of known T. cruzi lineages, and to improve and simplify the detection of infection in neonatal blood. We found that the two primers, Tcz1/Tcz2 and Diaz1/Diaz2, which target the 195-basepair satellite repeat, detected all parasitic lineages with the same sensitivity. However, the intensity of the amplicon was somewhat higher with Tcz1/Tcz2. For other tested primers (nuclear DNA primers BP1/BP2, O1/O2, Pon1/Pon2, and Tca1/Tca2 and kinetoplast DNA primers S35'/S36' and 121/122), either the intensity of amplicons varied according to T. cruzi lineages or the PCR assay was less sensitive. The use of the Tcz1/Tcz2 primers, which target a tandem repetitive sequence, requires a careful determination of the appropriate amount of Taq polymerase to avoid the formation of smears and multiple amplicon bands. The Tcz1/Tcz2 primers resulted in an intense 200-basepair amplicon with DNA extracted from blood equivalent to 0.02 parasites per assay when used with a simple DNA extraction method and of a low amount of Taq polymerase from a standard PCR kit. To better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested by parasitologic methods. The reliability of our PCR test was demonstrated, since all the 18 blood samples from newborns with congenital T. cruzi infection were positive, whereas the remaining samples (30 from control newborns of uninfected mothers and 262 of 263 from babies born to infected mothers) were negative. Since our PCR method is simple, reliable, robust, and inexpensive, it appears suitable for the detection of T. cruzi infection in neonatal blood, even in laboratories that are not equipped for performing the PCR.

Animals↗
PubMed1997

De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3' CCA sequence.

The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3' tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3' OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3' end of the retroplasmid transcript, which ends with a 3' CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3' CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3' end of the transcript. Single nucleotide changes in the 3' CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5' end of the cDNA during de novo initiation or to the 3' end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3' CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3' CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.

Base Composition↗
PubMed1994

DNA sequencing directly from a mixture using terminal-base-selective primers.

DNA cloning is often used to select and amplify one DNA species from a mixture. However, the cloning process is complex and labor-intensive. We have developed a new two-step method for DNA sequencing directly from a mixture. The first is the introduction of a known oligonucleotide (common part) into the terminus of unknown DNA by ligation. The second is selective DNA sequencing using primers with two additional nucleotides at the 3' terminus in addition to the common part (terminal-base-selective primers). The primers work only for templates on which the primers perfectly hybridized. This method was found to be effective for the HindIII digestion products of lambda phage.

Bacteriophage lambda↗
PubMed1996

Generation of sequence signatures from DNA amplification fingerprints with mini-hairpin and microsatellite primers.

DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationships between plant tax at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure and DAF efficiency. Mini-hairpin primers with short arbitrary cores and primers complementary to simple sequence repeats present in microsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is a dual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproducibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such as primer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynodon) cultivars and detecting putatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.

DNA Fingerprinting↗
PubMed2005

Amplification of human DNA by primers targeted to Leishmania kinetoplast DNA and post-genome considerations in the detection of parasites by a polymerase chain reaction.

We evaluated the Leishmania Viannia-specific primers B1-B2 to detect Leishmania in normal skin and peripheral blood monocytes of patients with active cutaneous leishmaniasis. Southern blotting and sequencing of polymerase chain reaction (PCR) products confirmed the specificity of kinetoplast DNA (kDNA) amplification from tissue fluid from healthy skin, whereas the PCR with monocytes also amplified a human sequence of a size similar (718 basepairs) to the expected kDNA product (750 basepairs), resulting in false-positive results. Although B1 was not homologous to any human DNA sequence, B2 showed homology to a human chromosome 2 intergenic region (AC010878) at positions 35,881-36,599, which are spaced 718 nucleotides apart. Amplification of the human artifact from monocyte DNA was confirmed using the primer B2 alone. Examination of other primers reported for the PCR of kDNA from various species of Leishmania showed that six of seven were homologous to human DNA sequences. These findings underscore the importance of exploiting sequencing, bioinformatics, and DNA probes to refine molecular amplification techniques and to validate the performance of primers when used for new applications.

Animals↗
PubMed2006

The mechanism of DNA replication primer synthesis by RNA polymerase.

RNA primers for DNA replication are usually synthesized by specialized enzymes, the primases. However, some replication systems have evolved to use cellular DNA-dependent RNA polymerase for primer synthesis. The main requirement for the replication primer, an exposed RNA 3' end annealed to the DNA template, is not compatible with known conformations of the transcription elongation complex, raising a question of how the priming is achieved. Here we show that a previously unrecognized kind of transcription complex is formed during RNA polymerase-catalysed synthesis of the M13 bacteriophage replication primer. The complex contains an overextended RNA-DNA hybrid bound in the RNA-polymerase trough that is normally occupied by downstream double-stranded DNA, thus leaving the 3' end of the RNA available for interaction with DNA polymerase. Transcription complexes with similar topology may prime the replication of other bacterial mobile elements and may regulate transcription elongation under conditions that favour the formation of an extended RNA-DNA hybrid.

Bacteriophage M13↗
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↗