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At least 55 records · Page 3Linked to original sources
PubMed2000

Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3' terminus to reduce false-positive signals.

A reverse transcription PCR (RT-PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3' terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 degrees C or 45 degrees C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 degrees C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene.

3' Untranslated Regions↗
PubMed1998

Phi 29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates.

A 44 kDa C-terminal fragment of phi 29 DNA polymerase has been separately expressed and purified from Escherichia coli cells. As expected, the truncated protein lacked the 3'-5' exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of phi 29 DNA polymerase. On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn2+ as metal activator, although the catalytic efficiency was greatly reduced with respect to that of the complete enzyme. Moreover, and in contrast to the high processivity exhibited by phi 29 DNA polymerase (> 70 kb), polymerization by its C-terminal domain was completely distributive. All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization. Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of phi 29 DNA replication. We conclude that the C-terminal portion of phi 29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis. These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more specific functions (TP-priming, high processivity and strand-displacement) inherent to phi 29 DNA polymerase.

Bacillus Phages↗
PubMed1996

Formation of chimeric DNA primer extension products by template switching onto an annealed downstream oligonucleotide.

Oligodeoxynucleotide sequences are described that anneal to a template downstream of a priming site. During polymerase-catalyzed extension of the primer, the extending primer shifts from the original template to a segment of the annealed oligonucleotide that acts as an alternative template. The resulting chimeric extended primer has one segment that is complementary to the template and a second segment that is complementary to the oligonucleotide. The influence of the sequence elements of the oligonucleotide and the reaction conditions on template switching have been explored. The sequence requirements for template switching are compared to those for transposon excision.

Base Sequence↗
PubMed1994

Detection of rye chromosome 2R using the polymerase chain reaction and sequence-specific DNA primers.

Sequences derived from a rye gamma secalin gene were used as primers in polymerase chain reactions using DNA obtained from a series of wheat and triticale genetic stocks. A 473-bp fragment, the predicted size based on the distance between the selected primers, was found only in rye, triticales, and wheat lines carrying rye chromosome 2RS. Use of triticale lines with various wheat chromosome substitutions confirmed the chromosomal origin of the rye-specific marker. The presence of the 473-bp PCR product was always associated with the production of 75K secalins in grain samples. Thus, the primer sequences, and the clone of origin (pSC503), were both derived from the SEC-2 locus of rye chromosome 2RS.

Base Sequence↗
PubMed1999

Effects of gel material on fluorescence lifetime detection of dyes and dye-labeled DNA primers in capillary electrophoresis.

Investigations of fluorescence lifetimes of the dye 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. The dependence of fluorescence lifetime on gel matrix and on experimental conditions was studied in order to identify which factors may be important for optimization of multiplex fluorescence lifetime detection. Lifetimes were determined in both batch solution and on-the-fly, on-column in CE. Results show that lifetimes of the primer-attached dyes remain constant in gels of different composition. Additionally, multiexponential fluorescence decays are observed for primer-attached dyes in batch solutions of the cellulose sieving buffers but are reduced to monoexponential decays when measured on-the-fly, on-column in CE. Lifetime detectability can be improved by addition of an organic modifier to the gel matrix.

Acrylic Resins↗
PubMed2004

Microflora ecology of the chicken intestine using 16S ribosomal DNA primers.

The microflora in the gastrointestinal tract of broiler chickens influences digestion, health, and wellbeing. Analysis of chicken gut microflora has been mainly by culture-based methods. Studies using these techniques have been useful for identification and analysis of specific groups of bacteria, however, the use of enrichment medium precludes even relative quantitation of bacterial species. Recent advances in ribosomal DNA-based molecular techniques make it possible to identify different bacterial populations in environmental samples without cultivation. In this study, the intestinal microflora was examined using 16S ribosomal DNA (rDNA) targeted probes from bacterial DNA isolated from intestinal and cecal contents of chickens at 4, 14, and 25 d of age. The ribosomal gene sequence was amplified using PCR with universal primers to determine total bacterial DNA and specific primers directed at 6 bacterial species: Lactobacillus, Bifidobacterium, Salmonella, Campylobacter, Escherichia coli, and Clostridium. The use of universal primers extends these methods to allow determination of relative proportions of different bacterial species. The results indicated that in young chicks the major species present in the small intestines and ceca was Lactobacilli, with a Bifidobacteria population becoming more dominant in the ceca at older age. Clostridium was detected in some segments of the small intestine in young chicks. In older chickens, Salmonella, Campylobacter, and E. coli species were found in the ceca. This study has demonstrated the use of molecular techniques for determining relative proportions of bacterial species and monitoring pathogens in the chick gastrointestinal tract.

Aging↗
PubMed1997

On-the-fly fluorescence lifetime detection of labeled DNA primers.

The first application of frequency-domain, on-the-fly fluorescence lifetime detection to the detection, identification and resolution of fluorescent labeled oligonucleotide primers in capillary electrophoresis is presented. These studies, which are directed towards the development of four-decay DNA sequencing, employed two common sequencing primers, SP6 and M13/pUC. The primers were each tagged by a derivative of either fluorescein or BODIPY. Fluorescence emission maxima and lifetimes of the free dyes and the labeled primers were determined both in batch mode and on-the-fly in capillary electrophoresis. Fluorescence intensity and lifetime electropherograms were extracted from dynamic lifetime data that were acquired at 0.1-s intervals during the CE separation. Lifetimes were recovered using either conventional non-linear least-squares analysis or the self-modeling maximum entropy method, which does not require prior knowledge of the system. Based on migration time and fluorescence lifetime, peaks could be detected and identified, and co-eluting peaks could be resolved. Interference from background impurities and scattered light was greatly reduced by the combination of physical separation and lifetime resolution.

DNA Primers↗
PubMed1996

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.

Base Sequence↗
PubMed1985

Molecular cloning of potato spindle tuber viroid (PSTV) cDNA synthesized by enzymatic elongation of PSTV-specific DNA primers: a general strategy for viroid cloning.

Different cDNAs were synthesized by primer extension from the RNA of the severe strain KF 440 of potato spindle tuber viroid (PSTV) with the aid of reverse transcriptase using three PSTV-specific DNA molecules as primers. The cDNAs were made double-stranded and cloned into plasmid pBR 322. Various overlapping subgenomic DNA fragments were prepared from these clones and recombined in two different ways. In both cases a PSTV DNA copy was obtained which represented the entire PSTV RNA genome. The sequence of the DNA of one of the resulting full-length clones was identical with the original PSTV isolate, whereas the other clone showed one nucleotide change. On the basis of these results the advantages and problems of different strategies for the molecular cloning of the circular viroid RNA genome are discussed.

Base Sequence↗
PubMed2004

Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers.

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Adenosine Triphosphate↗
PubMed1996

Structural variation among retroviral primer-DNA junctions: solution structure of the HIV-1 (-)-strand Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC).

The three-dimensional solution structure of the hybrid-chimeric duplex r(gcca)d(CTGC).d(GCAGTGGC) has been determined by two-dimensional NMR, restrained molecular dynamics (rMD), and NOE back-calculation methods. This chimera, consisting of a chimeric RNA-DNA strand and its complementary DNA strand, is formed after priming (-)-strand DNA synthesis by tRNA(Lys3) and subsequent (+)-strand DNA synthesis by reverse transcriptase and is an obligatory intermediate in the formation of double-stranded DNA prior to HIV-1 retrovirus integration. The duplex consists of two different types of double helix: a hybrid form (H-form) and a B-form structure connected by a junction. It is chemically similar to several other Okazaki fragments whose structures have been previously determined in our laboratory. However, some structural parameters are not the same and were found to be sequence dependent. In particular, the sugar conformations at the DNA base pair proximal to the hybrid segment vary from O4'-endo to C2'-endo depending on the base composition. The position of the transition from the relatively wide groove of H-form to the narrow groove of B-form is also sequence dependent, occurring either exactly at the RNA-DNA junction or within the purely DNA segment of the chimera-as is the case in the structure of the present HIV-1 (-)-strand primer. This structural change produces a kink at the DNA-DNA step adjacent to the RNA-DNA junction in the HIV-1 (-)-strand primer. The sequence dependence of structures of RNA-DNA chimeric duplexes may be responsible for the variable cleavage pattern of different Okazaki fragments by reverse transcriptase RNase H.

Base Composition↗
PubMed1994

A universal nucleoside for use at ambiguous sites in DNA primers.

A non-discriminatory base analogue, or universal base, would be an invaluable component of oligonucleotide probes and primers for solving the design problems that arise as a result of the degeneracy of the genetic code, or when only fragmentary peptide sequence data are available. We have designed an alternative to previous universal nucleoside candidates, a new analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (designated M; Fig. 1), which maximizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex. Oligonucleotides containing M at several sites were used as primers for sequencing and the polymerase chain reaction. The sequencing primer d(5'-CGT AAM CAM AAM ACM AT-3') is as effective as the exact match d(5'-CGT AAT CAG AAA ACA AT-3'). It is also possible to sequence using a primer containing M at several contiguous positions, for example d(5'-CGT AAT MMM MMM MMM AT-3'). Melting curves show that duplexes formed on hybridization of the sequences d(5'-CCT TTT TMT TTT TGG-3') and d(5'-CCA AAA AXA AAA AGG-3'), where X is A, C, G or T, melted at a lower temperature than the corresponding duplexes containing only d(A.T) and d(C.G) base pairs, but showing little variation among different X bases (Tm range 3 degrees C).

Animals↗
PubMed1993

Genetic characterization of six parasitic protozoa: parity between random-primer DNA typing and multilocus enzyme electrophoresis.

We have assayed genetic polymorphisms in several species of parasitic protozoa by means of random amplified polymorphic DNA (RAPD). One goal was to ascertain the suitability of RAPD markers for investigating genetic and evolutionary problems, particularly in organisms, such as the parasitic protozoa, unsuitable for traditional methods of genetic analysis. Another goal was to test certain hypotheses concerning Trypanosoma cruzi, and other protozoa, that have been established by multilocus enzyme electrophoresis. The RAPD results corroborate the hypothesis that the population structure of T. cruzi is clonal and yield a phylogeny of the clonal lineages in agreement with the one obtained by enzyme electrophoresis. This parity between the two sets of results confirms that RAPD markers are reliable genetic markers. The RAPD markers are also suitable for reconstructing species phylogenies and as diagnostic characters of species and subspecific lineages. The number of DNA polymorphisms that can be detected by the RAPD method seems virtually unlimited, since the number of primers can be increased effectively at will. The RAPD method is well suited for investigating genetic and evolutionary questions in certain organisms, because it is cost effective and demands no previous genetic knowledge about the organism.

Animals↗
PubMed1982

A warning on the use of synthetic DNA primers for initiation of reverse transcription on RNA templates: unexpected initiation at a mismatched nucleotide.

A synthetic 13-mer oligodeoxynucleotide designed to specifically initiate on lactic dehydrogenase (LDH) mRNA was shown to prime with high efficiency the synthesis of DNA complementary to the last 133 nucleotides of mitochondrial 16S ribosomal RNA on a mouse liver total poly(A)+ RNA template. One especially interesting feature of this reaction was that reverse transcriptase has been able to initiate at the 3' terminal C residue of the primer despite the fact that it was facing an A. This observation therefore establishes the ability of reverse transcriptase to initiate directly on a mismatched nucleotide.

Animals↗
PubMedDate not supplied

Cloning cDNAs corresponding to the 5' end of messenger RNAs using specific DNA primers.

A method is described to clone cDNAs corresponding to the 5' end of specific mRNAs which are absent from cDNA libraries. A segment is excised from the 5' end of a previously cloned truncated cDNA and hybridized to total polyadenylated RNA. The hybridized primer is extended using reverse transcriptase, tailed with deoxycytidine, and the second strand synthesized using oligo (dG)10-16 as a primer. The primer-extended double-stranded cDNA is cloned into pBR322 by dC-dG homopolymeric tailing. This method has been successful for cloning a 398 bp cDNA fragment corresponding to the entire 5' end of rat pancreatic elastase I mRNA, a prominent pancreatic mRNA, and a 510 bp fragment encoding the 5' end of kallikrein mRNA, a lower abundant pancreatic mRNA.

Animals↗