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Results for “Polymerase Chain Reaction”

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PubMed1994

Absence of Helicobacter pylori in subgingival samples determined by polymerase chain reaction.

The polymerase chain reaction was used for the detection of Helicobacter pylori from subgingival plaque in 336 periodontitis patients. A pair of primers derived from the H. pylori urease gene A served to amplify a targeted 411-bp fragment of genomic DNA. This technique permitted the detection of as few as 60 H. pylori cells. Paper point samples from 3 deep periodontal pockets per patient were immersed in 1 ml of phosphate-buffered saline or distilled water, DNA was solubilized by detergent/protease method, 3.7 microliters or 37 microliters of lysate supernatant was used as template, and the amplification product was analyzed in 1% agarose gel containing ethidium bromide. Each experiment included purified DNA and cell lysate of H. pylori as positive controls. The presence of bacteria in the sample was verified by a primer pair common to prokaryote 16S rRNA. The present study did not reveal the specific polymerase chain reaction amplification product characteristic of H. pylori. We conclude that periodontal pockets do not constitute a natural reservoir for H. pylori.

Adult↗

PubMed1998

Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction.

The polymerase chain reaction (PCR) has been established as a highly sensitive technique for detection of viral DNA or RNA. However, due to inherent limitations of PCR the amount of amplified product often does not correlate with the initial amount of template DNA. This is particularly true for PCR detection of viral infections that are characterized by low in vivo viral copy numbers in certain stages of the infection, such as human T-cell lymphotropic virus type 1 (HTLV-1) and simian T-cell lymphotropic virus type 1 (STLV-1). Therefore, we developed a quantitative competitive polymerase chain reaction (qcPCR) for detection of HTLV-1 and STLV-1 proviral DNA. The assay was optimized using an infectious HTLV-1 clone, ACH, HTLV-1 infected cell lines, MT-2.6 and HUT-102 and STLV-1 infected lines Kia and Matsu. Applicability of this system was demonstrated by determining HTLV-1 proviral load in peripheral blood mononuclear cells (PBMC) of human subjects with HTLV-1 associated diseases and an asymptomatic carrier as well as rabbits infected experimentally. This qcPCR method, the first designed specifically for HTLV-1 and STLV-1, will provide an important tool for pathogenesis studies of HTLV-1 and for evaluating the efficacy of antiviral drugs and vaccines against the viral infection using animal models.

Animals↗

PubMed1997

Quantitative analysis of mu and delta opioid receptor gene expression in rat brain and peripheral ganglia using competitive polymerase chain reaction.

Competitive polymerase chain reaction assays following reverse transcription have been developed for quantitative analysis of delta and mu opioid receptor gene expression. The assay was used to obtain quantitative measurements of mu and delta opioid receptor expression levels in different brain regions and sensory and sympathetic ganglia in the rat. The assays provide accurate estimates of the relative levels of receptor messenger RNAs by the inclusion in the assays of known amounts of internal standards with the same sequence, except for a small deletion, as the target complementary DNA. The amplification products of target and competitor can be distinguished by size, and their amounts measured by densitometry. Expression of mu and delta opioid receptor messenger RNAs in different regions of the rat brain, somatic and visceral sensory and sympathetic ganglia was investigated using this method. In the brain the highest density of delta receptor messenger RNA was detected in the olfactory bulb, followed by the striatum. The mu receptor was expressed at highest levels in the midbrain-hypothalamic region. All the sensory ganglia studied expressed both mu and delta opioid receptor messenger RNAs. In the nodose ganglion we observed the highest level of mu receptor messenger RNA of any structure studied; in the trigeminal ganglion the level was about 10 times lower than that in the nodose ganglion. Among the dorsal root ganglia, mu receptor messenger RNA density was highest in the lumbar region, followed by the thoracic and cervical regions. The sympathetic superior cervical ganglion expressed a very low level of mu message. Delta receptor messenger RNA was detected only in the sensory ganglia, at levels that were considerably lower than in the striatum. The reverse transcription polymerase chain reaction assay is quantitatively reliable for comparison of messenger RNA levels between different RNA extracts, and sensitive enough to permit the detection and assay of mu and delta opioid receptor gene expression in a single pair of sensory or autonomic ganglia from the rat.

Animals↗

PubMed1989

Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction.

The polymerase chain reaction was used to clone a full-length human methylmalonyl-CoA mutase cDNA from a human liver library by priming with sequences from the 5' end of a partial cDNA and sequences in the phage vector. The amino acid sequence predicted from the cDNA corresponds to the authentic amino acid sequences of peptide fragment from purified methylmalonyl-CoA mutase. The open reading frame of the cDNA encodes 742 amino acids (82,283 Da) comprising a 32 amino acid mitochondrial leader sequence and a mature protein of 710 amino acids (78,489 Da). The use of the polymerase chain reaction to "screen" the cDNA library represents a novel application of this technique. The full length will enable analysis of mutations underlying inherited methylmalonic acidemias caused by deficiency of the methylmalonyl-CoA mutase apoenzyme.

Amino Acid Sequence↗

PubMed1989

Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction.

The polymerase chain reaction was used to amplify a 188-base pair (bp) segment of the repetitive 195-bp nuclear DNA sequence of Trypanosoma cruzi that is the most abundant sequence in this organism. The reaction amplified this repetitive element in four T. cruzi isolates from widely separated geographic regions. No amplification of the 188-bp fragment occurred when DNAs extracted from Leishmania spp., African trypanosomes, or blood samples from mice and humans were used. Amplification of one-half of the DNA from a single T. cruzi parasite produced an amount of the 188-bp element that was readily visible in a gel stained with ethidium bromide. Hybridization of a radiolabeled probe to membrane-bound amplification products increased the sensitivity to a level at which 1/200 of the DNA in a single parasite could be detected. T. cruzi DNA was readily detected in DNA extracted from the abdominal contents of infected insect vectors reared in the laboratory. No parasite DNA was detected in the blood samples of two individuals known to be infected with T. cruzi, possibly because in such patients the number of circulating parasites are extremely low or because parasitemias are intermittent. These results represent a considerable increase in sensitivity over previously reported methods for the detection of T. cruzi infections. Polymerase chain reaction amplification can be used to evaluate large numbers of samples in a single day and thus should be useful in large-scale studies of the prevalence of T. cruzi in both insect vectors and mammalian hosts.

Animals↗

PubMed1991

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.

Base Sequence↗

PubMed2006

Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos.

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription-PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.

Animals↗

PubMed2003

Microfabricated device for DNA and RNA amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection.

We have developed a high-throughput microfabricated, reusable glass chip for the functional integration of reverse transcription (RT) and polymerase chain reaction (PCR) in a continuous-flow mode. The chip allows for selection of the number of amplification cycles. A single microchannel network was etched that defines four distinct zones, one for RT and three for PCR (denaturation, annealing, extension). The zone temperatures were controlled by placing the chip over four heating blocks. Samples and reagents for RT and PCR were pumped continuously through appropriate access holes. Outlet channels were etched after cycles 20, 25, 30, 35, and 40 for product collection. The surface-to-volume ratio for the PCR channel is 57 mm(-1) and the channel depth is 55 microm, both of which allow very rapid heat transfer. As a result, we were able to collect PCR product after 30 amplification cycles in only 6 min. Products were collected in 0.2-mL tubes and analyzed by agarose gel electrophoresis and ethidium bromide staining. We studied DNA and RNA amplification as a function of cycle number. The effect of the number of the initial DNA and RNA input molecules was studied in the range of 2.5 x 10(6) - 1.6 x 10(8) and 6.2 x 10(6) - 2 x 10(8), respectively. Successful amplification of a single-copy gene (beta-globin) from human genomic DNA was carried out. Furthermore, PCR was performed on three samples of DNA of different lengths (each of 2-microL reaction volume) flowing simultaneously in the chip, and the products were collected after various numbers of cycles. Reverse transcription was also carried out on four RNA samples (0.7-microL reaction volume) flowing simultaneously in the chip, followed by PCR amplification. Finally, we have demonstrated the concept of manually pumped injection and transport of the reaction mixture in continuous-flow PCR for the rapid generation of amplification products with minimal instrumentation. To our knowledge, this is the first report of a monolithic microdevice that integrates continuous-flow RT and PCR with cycle number selection.

DNA↗

PubMed1991

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction.

The polymerase chain reaction (PCR) in cerebrospinal fluid was compared with conventional bacteriology and an enzyme-linked immunosorbent assay (ELISA) for cerebrospinal fluid antibodies in the diagnosis of tuberculous meningitis (TBM). PCR was the most sensitive technique; it detected 15 (75%) of 20 cases of highly probable TBM (based on clinical features), 4 (57%) of 7 probable cases, and 3 (43%) of 7 possible cases. ELISA detected 11 (55%) of the highly probable cases and 2 each of the probable and possible cases. Culture was positive in only 4 of the highly probable cases. Among the controls (14 pyogenic meningitis, 3 aseptic meningitis, 34 other neurological disorders), 6 subjects tested early in the study (2 pyogenic meningitis, 4 other disorders) were PCR positive. Second DNA preparations from their stored cerebrospinal fluid samples were all PCR negative, suggesting that the false-positive results were due to cross-contamination. 18 PCR-positive TBM samples retested were all still PCR positive. The antibody ELISA was positive in 3 controls despite the use of a high cutoff value.

Bacteriological Techniques↗

PubMed2004

Systematic multiplex polymerase chain reaction and reverse transcription-polymerase chain reaction analyses of changes in copy number and expression of proto-oncogenes and tumor suppressor genes in cancer tissues and cell lines.

Systematic multiplex reverse transcription-polymerase chain reaction (SM RT-PCR) is distinguishable from other multiplex RT-PCR methods by (i) utilization of primers that amplify sequences that fall within a single exon of the genes, (ii) utilization of genomic DNA as a calibration standard, and (iii) optimized PCR conditions that allow amplification of bands of similar intensity using genomic DNA template. We previously developed the human experimental systems of 68 glycosyltransferase genes, 39 Hox genes, and 26 integrin subunit genes, and analyzed the expression of those genes in human adult tissues. Here we report the establishment of an SM RT-PCR system of proto-oncogenes and tumor suppressor genes and the analysis of gene expression in human cancer tissues and cell lines. We also demonstrate that the SM RT-PCR system, which was developed for cDNA expression analysis, could also be used successfully for more exquisite analysis of copy number changes in genomic DNA. We observed a decrease in band intensity of HRAS, TP73, CDKN2A, and CDKN2B genes in most of the breast and prostate cancer cell lines examined. The decrease in copy number of HRAS proto-oncogene leads us to suspect the presence of tumor suppressor genes in the vicinity of this gene on chromosome 11p15.5.

Cell Line, Tumor↗

PubMed1995

Detection of Chlamydia trachomatis by ligase chain reaction compared with polymerase chain reaction and cell culture in urogenital specimens.

OBJECTIVE: The aim of this study was to evaluate the newly developed ligase chain reaction (LCR) assay for the detection of Chlamydia trachomatis in urogenital specimens using cell culture and Amplicor PCR for comparison. SUBJECTS: Two hundred and eighty patients attending hospital or urban STD clinics (high-risk population, 62 men and 84 women) and obstetric/gynaecology clinics (low-risk population, 134 women) in Bordeaux, France. METHODS: Specimens from men were tested with LCR on urethral swabs and urine, with Amplicor or urine, with cell culture on urethral swabs. Specimens from women were tested with LCR, Amplicor and cell culture on endocervical swabs and with LCR on urine. When the three methods generated different results, the LCR and Amplicor tests were repeated on the remaining samples. Samples with discordant LCR and Amplicor results and a negative culture were further analysed by major outer membrane protein gene omp1-PCR. RESULTS: After analysis of discrepant results, the overall prevalence was 7.5% (21/280) calculated on the basis of an expanded "gold standard" defined as culture positive or LCR plus Amplicor positive or omp1-PCR positive for discrepant results between LCR and Amplicor tests. Of the 21, 20 were detected by LCR, 17 by Amplicor and culture. The specificity of LCR and Amplicor was 99.6%. CONCLUSION: The LCR Chlamydia trachomatis test is a highly sensitive nonculture technique and a good alternative test for the detection of chlamydial infections.

Bacteriological Techniques↗

PubMed1989

The polymerase chain reaction.

The polymerase chain reaction (PCR) is a novel technique that amplifies specific sequences with remarkable efficiency. Repeated cycles of denaturation, primer annealling and extension carried out with the heat stable enzyme, Taq polymerase, leads to exponential increases in the target DNA sequences. The technique has been widely applied in immunology to define HLA polymorphism, T-cell receptor and immunoglobulin diversity, pathogen detection, quantification of lymphokines and lymphoma or leukaemia detection. In addition, it has been very widely applied in all areas of molecular biology and human genetics. Because of its simplicity and sensitivity, PCR represents a major technical development for molecular biologists and will continue to have a great impact on molecular immunology. This review by John Bell describes the basic PCR technique and some variations on it and briefly explores their uses in molecular immunology.

DNA↗

PubMedDate not supplied

A comparison of thermocycling devices for automating the polymerase chain reaction.

The polymerase chain reaction (PCR) has become a valuable tool in the fields of cellular and virological research. This paper presents a performance comparison of four devices (three commercial and one prototype) designed to supply the thermic cycle central to the PCR procedure. Results of investigations using a thermocouple revealed that the greatest heating rate and shortest cycle time was attained by the prototype device which used waterbaths as the conducting medium. Dry-block based systems, which heated and cooled a single thermal reservoir, provided generally smaller and more sophisticated machines, but two such devices produced varying results when subjected to experimental analysis. Before purchasing, it is recommended that comparative trials are conducted using differing types of machines, since performance may vary according to individual reactions executed.

Equipment Design↗

PubMed1990

Detection of caprine arthritis-encephalitis- and maedi-visna viruses using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to demonstrate proviral DNA of lentiviruses of small ruminants in cultured cells. Primers for the Taq polymerase were selected in the GAG gene of Icelandic maedi-visna virus and POL gene of caprine arthritis-encephalitis (CAE) virus. Using PCR, proviral DNA of CAE virus was detected at 1 day post infection, 4 days before viral protein could be demonstrated using a sensitive immunoblotting protocol and 6 days before the appearance of syncytia. Primers derived from the published sequence of CAE virus successfully primed for the synthesis of homologous virus and Icelandic maedi-visna virus but not for maedi-visna virus isolated in The Netherlands. In contrast, primers derived from the GAG region of Icelandic maedi-visna virus allowed the amplification of DNA of homologous virus, maedi-visna virus isolated in The Netherlands as well as CAE virus.

Amino Acid Sequence↗

PubMed2004

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction.

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.

DNA, Mitochondrial↗

PubMed1990

DNA amplification by the polymerase chain reaction.

The polymerase chain reaction (PCR) is a technique involving enzymatic amplification of nucleic acid sequences via repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension. PCR has revolutionized the practice of DNA technology as it allows virtually any nucleic acid sequence to be readily generated in vitro in relatively great abundance, so that subsequent analyses are not confounded by the presence of other DNA fragments or a lack of material with which to work. PCR also enables the sequence of individual DNA fragments to be altered. The method has advantages over conventional procedures for DNA cloning and analysis in many circumstances because it is faster, simpler, and more flexible. The total range and number of applications that have evolved in the short time since the first report of PCR are enormous. This review describes some of the history of PCR, the principle of the method, practical considerations for performing PCR, and a variety of applications.

Gene Amplification↗

PubMedDate not supplied

Differentiation of pathogenic and non-pathogenic leptospires by means of the polymerase chain reaction.

A polymerase chain reaction was carried out to detect pathogenic leptospires isolated from animals and humans in Argentina. A double set of primers (G1/G2, B64-I/B64-II), described before, were used to amplify by PCR a DNA fragment from serogroups belonging to Leptospira interrogans but did not allow to detect saprophytic strains isolated from soil and water (L. biflexa). This fact represents an advantage since it makes possible the differentiation of pathogenic from non-pathogenic leptospires in cultures. The sensitivity of this assay has been determined, allowing to detect just only 10 leptospires in the reaction tube. Those sets of primers generated either a 285 bp or 360 bp fragment, depending on the pathogenic strain.

Animals↗

PubMedDate not supplied

Detection of Borrelia burgdorferi DNA in human skin biopsies and dog synovial fluid by the polymerase chain reaction.

The polymerase chain reaction was used to amplify DNA sequences of the etiologic agent of Lyme disease, Borrelia burgdorferi, and was applied to the detection of the spirochete in humans and dogs. Oligonucleotide primers used in the reaction flank a 244-base-pair representing part of the variable region V4 of the B. burgdorferi 16S rRNA from biopsies of patients with acrodermatitis, and in synovial fluid from a dog with arthritis. These data suggest the presence of the disease in our state.

Animals↗