Computing by polymerase chain reaction

A mathematical notation is introduced to represent, at a symbolic level, different mechanisms of DNA recombination, and a 'PCR lemma' is proven by analytically describing the combinatorial properties of the polymerase chain reaction process. This approach led to the discovery of novel techniques, based on a form of PCR which we called cross pairing PCR (briefly XPCR). They were mathematically analyzed and already experimentally proven in different contexts, such as DNA extraction and recombination. Thus, a mathematical analysis of standard methodologies may highlight novel mechanisms of DNA recombination and this can provide new technologies for DNA manipulation.     

Diagnosis of nocardiosis by polymerase chain reaction: an experimental study in mice

In this study, we have established a sensitive semi-nested polymerase chain reaction (PCR) for detection of Nocardia in clinical specimens by first amplifying a 422 bp DNA fragment from the groEL gene, followed by a second amplification of 342 bp DNA by targeting sequences internal to the first amplicon. The semi-nested PCR was evaluated in a murine model of nocardiosis for detection of Nocardia in blood and visceral organs. Healthy BALB/c mice were intravenously infected with 0.2 ml suspension of 2.9 × 10⁵/ml cfu of Nocardia asteroides and N. farcinica. Viable counts and semi-nested PCR were performed post-infection with samples of blood as well as lung, liver, spleen, kidney and brain at 5 minutes, 3 h, and then every 24 h for 3 days. Of the 20 blood samples tested, 15 (75%) were Nocardia positive by culture and 19 (95%) were positive by semi-nested PCR. Likewise, in case of N. asteroides infection, 46% organ samples were positive by culture and 58% by semi-nested PCR. The positivity of organ samples was higher with N. farcinica, 60% by culture and 72% by PCR, which may be attributed to its increased virulence as compared to N. asteroides. These results demonstrate that semi-nested PCR is a rapid and sensitive method for detection of Nocardia in blood and different visceral organs. The diagnostic application of this method provides an additional advantage over culture techniques, as PCR can also detect L-forms of Nocardia in clinical specimens, which otherwise fail to grow on routine isolation medium.     

The detection of transgenic animals using a polymerase chain reaction in situ

The technique for detecting both foreign and host specific DNA sequences inside nuclei and chromosomes of single cells of transgenic mice with the help of polymerase chain reaction (PCR) in situ is described. The mouse preimplantation and postimplantation embryonic and adult cells were studied. The methodology is described in detail with particular attention to the optimization of composition of reaction mixture, kind of fixation and preliminary denaturation of target DNA. The reaction takes only several hours and needs no sophisticated equipment.     

In situ polymerase chain reaction and cycling primed in situ amplification: improvements and adaptations

 Ethanol fixation combined with microwave pretreatment allows rapid and simple detection of signals produced by cycling primed in situ (PRINS) amplification, which uses a single primer, and in situ polymerase chain reaction (ISPCR) in intact cells. After thermal cycling, signals remain as discrete subnuclear spots in the region of amplification and are clearly distinguishable from non-specific background labelling. These methods are applicable to routine blood smears, even after Giemsa staining or immunocytochemistry, and cellular morphology is retained. Chromosome enumeration by cycling PRINS is demonstrated using primers for repeat DNA sequences, whilst single copy sequence detection is demonstrated using bcl-2, CFTR and chromosome 21 specific primer pairs in ISPCR. We show that ethanol fixation supports efficient extension of cycling PRINS products to approximately 550 bp using up to 70 rounds of thermal cycling. Accepted: 15 February 1999     

Detection of Toxoplasma gondii by polymerase chain reaction in sera of acutely infected mice

The presence of Toxoplasma gondii DNA was detected in sera of acutely infected mice by polymerase chain reaction. Adult mice were inoculated intraperitoneally with 5 x 10(3) T. gondii RH strain tachyzoites. Five mice were killed every 3 hr from 3 to 21 hr post infection (PI) and every day from 1 to 7 days PI. Toxoplasma gondii DNA was first detected in 60% of the infected mice 18 hr PI and in 100% of the animals 21 hr PI and from 1 to 7 days PI. No mice survived longer than 7 days.     

Detection and localization of HIV-1 DNA in renal tissues by in situ polymerase chain reaction

The localization of HIV-1 DNA in renal tissues is critically important for understanding pathogenesis of HIV-associated nephropathy (HIVAN), but the clarification has been technically challenging. We applied in situ polymerase chain reaction (IS-PCR) to human renal tissues to demonstrate viral entry into the renal epithelial cells in vivo. To test the specificity of this method and to determine the cell types infected, we used IS-PCR followed by in situ hybridization (ISH) and IS-PCR followed by immunohistochemistry and histochemical counterstains. Brief 2 hour fixation in 4% paraformaldehyde had 92.9% sensitivity and 100% specificity for detection of viral DNA in renal biopsies of HIVAN patients, compared to 70.8% sensitivity and 66.7% specificity in renal biopsies fixed overnight in 10% formalin. Under optimized conditions, the only signals detectable in HIV-1 seronegative cases were false positives attributable to renal tubular apoptosis. In HIVAN cases, positive signal was observed in podocytes, parietal cells, renal tubular cells, and interstitial leukocytes. Immunohistochemical co-labeling for pan-T cell and macrophage markers revealed that the interstitial leukocytes with positivity for HIV-1 DNA included both T cells and macrophages. Application of ISH after IS-PCR showed the same distribution of signal as observed using IS-PCR alone, confirming the specificity of the technique. IS-PCR is a powerful technique to detect viral DNA in human tissue sections, but requires proper use of negative controls to set optimal fixation, protein digestion, and amplification conditions.     

Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ polymerase chain reaction

Classical in situ hybridization (ISH) with biotinylated probes makes it possible to detect and localize human papillomavirus (HPV) nucleic acid sequences in cytological and histological materials. This method is however of limited value in the detection of a few copies of the virus. Moreover the specificity of such a technique is not always convincing when ISH signals are small and/or of low intensity. Recently, much attention has been focused on the utility of the in vitro polymerase chain reaction (PCR) and especially on PCR-single strand conformation polymorphism (SSCP) to amplify small amounts of viral DNA with accurate hybrid specificity. But the latter method requires nucleic acid extraction and tissue destruction. Thus, correlation between the PCR results and histological findings is not possible. Hence, the aim of our current study was to apply to HeLa cells and cervical formalin-fixed and paraffin-embedded biopsies, a novel procedure of ISH signal amplification, the catalyzed signal amplification (CSA). Such a procedure is based on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probed target. The biotin accumulation is then detected with streptavidin peroxidase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification successfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a method was found simpler and faster than in situ PCR and tissue morphology was better preserved.     

Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction

AIMS: To carry out a rapid and reliable identification of bacterial diversity in the oyster Crassostrea gigas from Todos Santos Bay, México, in the current study we applied the molecular techniques of fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In order to reach this goal, genus and group-specific oligonucleotides targeted to 16S rDNA/rRNA were used. METHODS AND RESULTS: Oysters were collected and different tissues were analysed by means of culture-independent methodologies. In the digestive glands and gonads gamma-Proteobacteria and Gram-positive bacteria with a low G+C content, were identified as metabolically active by FISH. In the oyster gills a higher active diversity was observed, including Gram-positive bacteria with a low and high G+C content, members of the Cytophaga/Flavobacterium cluster and gamma-Proteobacteria. Consistent with FISH analysis, the amplification of 16S rDNA genes fragments with genus and group-specific oligonucleotides confirmed the presence of the same groups, as well as members of the alpha- and beta-Proteobacterias, Pseudomonas spp. and Bacillus spp. CONCLUSIONS: The combination of accurate and very easy-to-apply molecular methods allowed us to carry out a rapid screening of high bacterial diversity in oysters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first report about bacterial diversity in oyster tissues analysed by FISH and PCR, without using culture-dependent methods and allowed us to determine the phylogenetic diversity of the bacterial communities present in oyster cultures, including bacteria with and without metabolic activity, as well as uncultivable cells, which are generally underestimated by traditional identification.     

Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction

The use of in situ techniques to detect DNA and RNA sequences has proven to be an invaluable technique with paraffin-embedded tissue. Advances in non-radioactive detection systems have further made these procedures shorter and safer. We report the detection of Trypanosoma cruzi, the causative agent of Chagas disease, via indirect and direct in situ polymerace chain reaction within paraffin-embedded murine cardiac tissue sections. The presence of three T. cruzi specific DNA sequences were evaluated: a 122 base pair (bp) sequence localized within the minicircle network, a 188 bp satellite nuclear repetitive sequence and a 177 bp sequence that codes for a flagellar protein. In situ hybridization alone was sensitive enough to detect all three T. cruzi specific DNA sequences.     

Estimation of the reaction efficiency in polymerase chain reaction

Polymerase chain reaction (PCR) is largely used in molecular biology for increasing the copy number of a specific DNA fragment. The succession of 20 replication cycles makes it possible to multiply the quantity of the fragment of interest by a factor of 1 million. The PCR technique has revolutionized genomics research. Several quantification methodologies are available to determine the DNA replication efficiency of the reaction which is the probability of replication of a DNA molecule at a replication cycle. We elaborate a quantification procedure based on the exponential phase and the early saturation phase of PCR. The reaction efficiency is supposed to be constant in the exponential phase, and decreasing in the saturation phase. We propose to model the PCR amplification process by a branching process which starts as a Galton-Watson branching process followed by a size-dependent process. Using this stochastic modelling and the conditional least-squares estimation method, we infer the reaction efficiency from a single PCR trajectory.     

Detection of mycoplasma contaminations by the polymerase chain reaction

The polymerase chain reaction (PCR) has been used for the general detection ofMollicutes. 25Mycoplasma andAcholeplasma species were detected including important contaminants of cell cultures such asM. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1–2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.     

Chromosomal location of the 28S ribosomal RNA gene of channel catfish by in situ polymerase chain reaction

This study describes the use of the polymerase chain reaction for physical mapping of fish genes. A 287–base pair (bp) fragment of the 28S ribosomal RNA gene (28S rDNA) of channel catfish Ictalurus punctatus was isolated and sequenced with human-derived primers. The nucleotide (nt) sequence of this fragment was 20 bp shorter than that of the corresponding region of the human 28S rDNA. The gene was mapped to chromosomes of channel catfish by fluorescence in situ hybridization (FISH) and in situ polymerase chain reaction (ISPCR). A major locus and a minor locus of 28S rDNA were found on chromosomes of channel catfish. The major locus was associated with the active nucleolus organizer region (NOR) sites. The minor locus was highly resolved and not detectable by silver staining, suggesting that this locus was not involved in synthesis of ribosomal RNA and possessed fewer copies of 28S rDNA. Both loci contained GC-rich DNA elements that could be components of 28S rDNA repeated units. In this study, a potential method of comparative mapping of the channel catfish genome has been presented by using human-derived oligonucleotide sequences. These data demonstrate that ISPCR is highly specific and will be useful in physical mapping of fish genomes.     

Inhibitors of the polymerase chain reaction

The information on the applied aspects of the polymerase chain reaction (PCR) is updated. In particular, the main inhibitor of PCR, considerably decreasing the sensitivity of the method both at the lysis stage of the tested material and due to the degradation of the DNA matrix and primers and/or to the direct inhibition of the activity of DNA polymerase, are described. The compounds, most frequently distorting the course of the reaction while testing clinical blood samples, bioptic samples, sputum, etc., are characterized. Testing concrete clinical material with the use of PCR was shown to require differentiated approach both at the stage of choosing the adequate method for the preparation of samples and at all other stages, including, e.g., the corresponding DNA polymerases or at the stage of heating for decreasing endonuclease activity or for IgG denaturation. Information on the causes of false negative results of PCR and the variants of their elimination, useful under practical laboratory conditions, is given.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.