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PubMed1991

OSP: a computer program for choosing PCR and DNA sequencing primers.

OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA sequencing and the polymerase chain reaction (PCR). The user can specify (or use default) constraints for primer and amplified product lengths, %(G+C), (absolute or relative) melting temperatures, and primer 3' nucleotides. To help minimize nonspecific priming and primer secondary structure, OSP screens candidate primer sequences, using user-specifiable cutoffs, against potential base-pairing with a variety of sequences present in the reaction, including the primer itself, the other primer (for PCR), the amplified product, and any other sequences desired (e.g., repetitive element sequences in genomic templates, vector sequence in cloned templates, or other primer pair sequences in multiplexed PCR reactions). Base-pairing involving the primer 3' end is considered separately from base-pairing involving internal sequences. Primers meeting all constraints are ranked by a "combined score," a user-definable weighted sum of any of the above parameters. OSP is being routinely and extensively used to select sequencing primers for the Caenorhabditis elegans genome sequencing project and human genomic PCR primer pairs for the Washington University Genome Center mapping project, with success rates exceeding 96% and 81%, respectively. It is available for research purposes from the authors, at no cost, in both text output and interactive graphics (X windows) versions.

Animals↗
PubMed2002

Design and use of 16S ribosomal DNA-directed primers in competitive PCRs to enumerate proteolytic bacteria in the rumen.

Competitive Polymerase Chain Reaction primers were designed for Streptococcus, B. fibrisolvens, P. bryantii, Eubacterium sp., Prevotella, and a universal primer for the eubacteria. DNA was extracted from rumen contents collected from eight dairy cows fed four diets: adequate nitrogen, adequate nitrogen plus carbohydrate, low nitrogen, and low nitrogen plus carbohydrate. B. fibrisolvens was significantly higher on the adequate nitrogen plus carbohydrate and the low nitrogen plus carbohydrate diets compared with the other diets, while P. bryantii was significantly higher on the low nitrogen plus carbohydrate diet compared with the adequate nitrogen diet. The population of Eubacterium sp. was significantly lower on both the adequate nitrogen plus carbohydrate and low nitrogen plus carbohydrate diets. Streptococcus populations were significantly lower on the low nitrogen plus carbohydrate diet compared with all three other diets, whereas there were no significant differences in populations of Prevotella or total eubacteria on any of the diets.

Animal Feed↗
PubMed1977

Action of pancreatic DNase: requirements for activation of DNA as a template-primer for DNA polymerase.

Pancreatic DNase requires both Ca2+ and Mg2+ for its activity as measured by formation of an activated DNA template for in vitro DNA polymerase alpha assay and by the hyperchromic shift. Mn2+ can partially satisfy the Mg2+ requirement of the DNase for activation of DNA but the resulting template is only 50% as active in the DNA polymerase assay. When precautions are taken to avoid divalent ion contamination, pancreatic DNase is not active in the presence of Ca2+ or Mg2+ alone. analysis of the DNA by sucrose gradient centrifugation shows that only in the presence of Ca2+ plus Mg2+ or Mn2+ does pancreatic DNase produce extensive strand breaks in the DNA. The activated DNA template that yields maximal DNA polymerase activity is low molecular weight material of 30,000 to 50,000 daltons.

Animals↗
PubMed1986

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis.

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.

Base Sequence↗
PubMed2001

Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders.

Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1.3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider.

Aldehydes↗
PubMed1981

A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322.

Seven oligonucleotide primers complementary to the plasmid vector pBR322 at positions adjacent to five of the unique restriction endonuclease cleavage sites (EcoRI, HindIII, BamHI, SalI and PstI) have been chemically synthesized. The polarity of the primers is such that any DNA inserted at one or a combination of two of the above restriction sites may be sequenced by the chain termination method using one of the synthetic DNA primers. One of the primers for sequencing inserts at the PstI site of pBR322 is also complementary to the M13 phage vector designated bla6. This set of universal primers is useful for rapid sequence determination of DNA cloned into pBR322 or M13bla6.

Base Sequence↗
PubMed1986

Effects of exposure of DNA to methyl mercury on its activity as a template-primer for DNA polymerases.

A previous publication [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)] described the observation that double-stranded DNA which was briefly exposed to methyl mercury (MeHg) and purified to remove free methyl mercury was transcribed at a higher rate by RNA polymerase II from wheat germ. The specificity of this phenomenon has now been investigated by examining the activity of this MeHg-exposed DNA as a template-primer for DNA polymerases. DNA synthesis by the bacteriophage T4-induced DNA polymerase was higher with the MeHg-exposed DNA as a template-primer than with control DNA. In contrast, the rate of DNA synthesis by E. coli DNA polymerase I was lower with the MeHg-exposed DNA as template-primer. With both enzymes (as well as with RNA polymerase II), after denaturation of the MeHg-exposed and control DNAs the differences in template activity were either eliminated or markedly reduced. The enzymes are thus able to detect a MeHg-induced alteration in DNA. In contrast, circular dichroism, a physical method that is sensitive to conformational changes in DNA, did not detect any difference between the MeHg-exposed and control DNAs.

DNA↗
PubMedDate not supplied

[Design of primers to DNA of lactic acid bacteria].

Primers LP1-LP2 to the gene 16S rRNA have been developed, which permit to differentiate lactic acid bacteria: Lactobacillus plantarum, L. delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus. The strain-specific and species-specific differentiations are possible under different annealing temperature. Additional fragments, which are synthesized outside the framework of gene 16S rRNA reading, provide for the strain-specific type of differentiation, and the fragment F864 read in the gene 16S rRNA permits identifying L. plantarum.

DNA Primers↗
PubMed1994

Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuclease analysis of amplified DNA.

Primers for PCR were selected from a sequenced fragment of clone pL590, which contains a repetitive element present in the genome of Leptospira interrogans serovar hardjo type hardjoprajitno (M. L. Pacciarini, M. L. Savio, S. Tagliabue, and C. Rossi, J. Clin. Microbiol. 30:1243-1249, 1992). A specific DNA fragment was amplified from the genomic DNAs of serovar hardjo type hardjoprajitno and nine serovars also belonging to L. interrogans as a consequence of the spread of the same or a closely related repetitive element within this species (Pacciarini et al., J. Clin. Microbiol. 30:1243-1249, 1992). In addition, specific amplification was obtained from two Leptospira borgpetersenii serovars (tarassovi and hardjo type hardjobovis). Negative PCR results were observed with all of the other Leptospira serovars tested, including nonpathogenic ones (serovars patoc and andamana), another spirochete (Borrelia burgdorferi), bacteria commonly found in biological samples, and swine and bovine cell lines. Direct PCR on biological samples such as kidney samples demonstrated that preliminary isolation and culture of Leptospira cells are not required for efficient detection. Furthermore, digestion of the amplified DNA with the enzymes HinfI and DdeI yielded specific polymorphic patterns, allowing discrimination among the majority of the serovars. These methods were applied to 25 field isolates of serovar pomona, leading to the conclusion that they were suitable for the simple and rapid detection of L. interrogans and for serovar identification.

Animals↗
PubMed1999

Detection of human papilloma virus (HPV) genomes by the primed in situ (PRINS) labelling technique.

Primed in situ Labelling, a technique based on primer mediated DNA synthesis, has become a useful tool in cytogenetics, especially for chromosome mapping, banding and the investigation of sequence organization in fresh metaphase preparations. Its application in the routine surgical pathology laboratory has been hampered by the fact that the technique did not work on paraffin-embedded, formalin-fixed tissue. We investigated cervical biopsies (n = 20) with morphological signs of HPV infection and found that the PRINS method is at least as sensitive as a classical in situ hybridization assay for detecting HPV DNA in paraffin-embedded, formalin-fixed tissue. In all investigated cases (n = 20), HPV DNA was found by both methods. The PRINS method was able to demonstrate HPV DNA not only in superficial koilocytotic squamous cells but also in non-koilocytotic cells in the deeper spinous cell layers, and even in some basal cells. We describe an economical protocol using conventional consensus HPV oligonucleotide DNA primers. The described method is rapid (approximately 3 hours) and easy to perform for screening and subtyping HPV infection in the routine surgical pathology laboratory.

DNA Primers↗
PubMed1996

Co-operativity of hexamer ligation.

The SPEL-6 (sequential rimer elongation by ligation of 6-mers) procedure is based on the assembly of DNA primers by ligation of three or more hexamers taken from a library of 4096 hexamers. In this way, the synthesized primers enable DNA sequencing by primer walking. Ligation by both T4 DNA ligase and Rhodothermus marinus thermophilic DNA ligase is highly cooperative. Sequencing ladders obtained with 18-60-nucleotide (nt) primers (produced by ligation of three to ten hexamers using T4 DNA ligase) were all of high quality, with no spurious bands. R. marinus DNA ligase requires at least seven hexamers for successful primer synthesis. Long primers (up to 60 nt), which are easy to obtain, especially by automated ligation, offer a definite advantage in DNA priming in regions with pronounced secondary structure. Moreover, the SPEL-6 procedure for DNA sequencing reduces the sequencing effort manifold. An additional application of hexamer ligation is the detection of point mutants, as described here.

DNA Ligases↗
PubMed1994

Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity. Isolation of new antimutator and mutator DNA polymerases.

Polymerases in general share only a few regions of amino acid similarity. One of the most conserved regions, called motif A, has the sequence DXXSLYPSII or a similar sequence in many eukaryotic and viral DNA polymerases and in bacteriophage T4 DNA polymerase. We designed genetic techniques to isolate mutant T4 DNA polymerases with amino acid substitutions in this highly conserved motif. The mutant DNA polymerases differed from wild type T4 DNA polymerase in several ways. For one mutant DNA polymerase, the pyrophosphate analog, phosphonoacetic acid, was a potent inhibitor of DNA replication, and this mutant DNA polymerase replicated DNA with reduced fidelity. Another mutant DNA polymerase replicated DNA with increased accuracy, but this mutant DNA polymerase was less processive in primer extension reactions, and DNA replication required high concentrations of deoxynucleoside triphosphates. We provide evidence that indicates that all of these changes to DNA polymerase function are due to differences in how the mutant DNA polymerases partition between states active for DNA replication or exonucleolytic proofreading. These studies also provide further support for the hypothesis that the accuracy of DNA replication observed for DNA polymerases and 3'-->5' exonuclease activities (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol. Chem. 247, 7116-7122).

Amino Acid Sequence↗
PubMed1998

[Genetic variability of Trichinella spiralis Oven, 1835, and Trichinella pseudospiralis Garkavi, 1972, detected by polymerase chain reaction with random primers].

DNA polymorphisms in two parasitic nematode species, Trichinella spiralis Oven, 1835, and Trichinella pseudospiralis Garkavi, 1972, were revealed via random amplification of polymorphic DNA by the polymerase chain reaction (RAPD PCR). The diagnostic value of seven 10-bp oligonucleotide primers was evaluated, and the extent of the homology between the genomes of the two species was estimated. The intraspecific variation of RAPD markers was revealed in larvae of both species isolated from experimentally infected white rats. The variation was higher in larvae from nonlinear rats than in larvae from linear rats. When animals were infected with both Trichinella species simultaneously, "hybrid" progeny were obtained that had capsule that somewhat differed in shape from one characteristic of the parental species, T. spiralis. In RAPD spectra, the hybrids showed higher similarity of T. spiralis than to T. pseudospiralis. Intra- and interspecific differentiation, genome divergence, and factors inducing the intraspecific variation in Trichinella species are discussed.

Animals↗
PubMed1992

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.

Base Sequence↗
PubMed2006

NMR investigation of DNA primer-template models: structural insights into dislocation mutagenesis in DNA replication.

Slipped frameshift intermediates can occur when DNA polymerase slows or stalls at sites of DNA lesions. However, this phenomenon is much less common when unmodified DNA is replicated. In order to study the effect of templating bases on the alignment of primer-templates, NMR structural investigation has been performed on primer-template oligonucleotide models which mimic the situation that dNTP has just been incorporated opposite template. NMR evidence reveals the occurrence of misalignment when dGTP is incorporated opposite template T with a downstream nucleotide C. Depending on the template sequence, further extension of the primer can lead to realignment.

Base Pair Mismatch↗
PubMed1998

PCR-introduced loop structure as primer in DNA sequencing.

The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is formed by elution of the non-bound strand. Here, we show that both the immobilized and the eluted strand can be analyzed using conventional Sanger DNA sequencing and the novel pyrosequencing method as described previously. By using a stem-loop structure as a primer for DNA sequencing, the risk for mispriming is minimized.

Base Sequence↗