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Results for “Polymerase Chain Reaction”

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At least 19 recordsLinked to original sources
PubMed1995

Detection of Rickettsia tsutsugamushi specific DNA from the lymphocyte of patients by polymerase chain reaction.

Polymerase chain reaction was used to detect Rickettsia tsutsugamushi specific DNA from the lymphocytes of patients in the acute phase of the disease. The primers used in the PCR were based on the nucleotide sequences of gene encoding of the 56 kDa antigen of the Gilliam strain. The PCR product is a 78 bp fragment which can be hybridized by the 78 bp DNA probe of the Gilliam strain. Comparison of the results obtained by polymerase chain reaction, immunofluorescent assay and cell culture suggests that the polymerase chain reaction method is the most sensitive one among the three in diagnosis of scrub typhus.

Acute-Phase Reaction↗
PubMed1992

Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction.

Polymerase chain reaction was used to amplify the low copy number of two 16S ribosomal gene fragments from soil and sediment extracts. Total DNA for polymerase chain reaction was extracted from 1 g of seeded or unseeded samples by a rapid freeze-and-thaw method. Amplified DNA fragments can be detected in DNA fractions isolated from seeded soil containing less than 3 Escherichia coli cells and from seeded sediments containing less than 10 cells. This research demonstrated that coupling polymerase chain reaction to direct DNA extraction improves sensitivity by 1 and 2 orders of magnitude for sediments and soils, respectively. This technique could become a powerful tool for genetic ecology studies.

Bacteria↗
PubMed1989

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Autoanalysis↗
PubMed2004

Detection and quantification of circulating tumor cells in patients with esophageal cancer by real-time polymerase chain reaction.

Polymerase chain reaction (PCR) amplification was used for detecting circulating tumor cells. However, PCR was so sensitive that it detected a very low level of mRNA with no relevance to tumor cells. We analyzed the degree of micro-tumor spread in esophageal cancer patients using quantitative PCR. Samples were collected from 28 patients and 35 controls. Real-time quantitative PCR (LightCycler) was employed for the detection of carcinoembryonic antigen (CEA) and cytokeratin 20 (CK-20). In the CEA and CK-20 mRNA assays, 7 and 3 out of 28 patients, respectively, showed higher mRNA levels in peripheral blood than the normal range based on values of controls (mean+/-2SD). Eleven out of 19, 4 out of 14, and 2 out of 5 patients showed higher CEA mRNA levels in the samples from tumor drainage vein, costal bone marrow, and thoracic duct lymph, respectively. One of the 7 patients who showed higher CEA mRNA levels in pretreatment peripheral blood is currently free from disease. These findings reveal that quantitative PCR can discriminate high levels of cancer-specific expression from low levels of illegitimate expression in blood. They also suggest that the identification of circulating tumor cells by the CEA mRNA assay is a reliable means of predicting early recurrence.

Aged↗
PubMed2000

Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases.

The aim of this study was to detect point mutations in extended-spectrum beta-lactamase (ESBL) genes in a background of wild-type (non-ESBL-producing) bacteria using a highly sensitive and specific method developed for this purpose. The ligase detection reaction-polymerase chain reaction (LDR-PCR) method was used to test different ESBL-producing strains and clinical isolates for a specific point mutation in the bla SHV-ESBL gene (glycine to serine mutation at position 238) and was compared with the commercially available E test ESBL (AB Biodisk, Sweden). Nine of the 40 clinical isolates tested were positive for the bla SHV-ESBL point mutation when tested by the LDR-PCR method but negative when tested by the E test. In contrast to the E test or other molecular genetic tests, the LDR-PCR method is able to identify a single bacterium with a point mutation in a background of 100,000 wild-type (non-ESBL-producing) bacteria.

Amino Acid Substitution↗
PubMed1993

Identification of Rickettsia prowazekii using the polymerase chain reaction.

Polymerase chain reaction (PCR) was used to identify Rickettsia prowazekii, the etiologic agent of epidemic typhus. For the PCR, Thermus thermophilus thermostable DNA polymerase was applied with buffer containing a relatively low Mg2+ concentration (1.5-2 mM with dNTP's at 250 microM each). A primer pair used to amplify a 448-base-pair (bp) fragment of R. prowazekii genome was synthesized on the basis of the DNA sequence of gene rpa14/16, coding for a precursor of the mature polypeptides of molecular weight (Mr) 14,000 and/or 16,000 (16kD) from R. prowazekii strain E. For determining the specificity of the primer pair, purified genomic DNAs of 16 rickettsial and 10 other bacterial strains were used.

Animals↗
PubMed1992

Coamplified positive control detects inhibition of polymerase chain reactions.

Polymerase chain reactions (PCR) may fail to react because the substrate DNA is absent (true negative) or because of inhibition of specific amplification (false negative). The use of positive controls can increase confidence in negative PCR results by ruling out failure due to inhibition as a cause of the lack of amplification products. This report describes the construction and application of coamplified positive controls for herpes simplex virus and human herpesvirus 6 amplifications. Herpes simplex virus and human herpesvirus 6 PCR products were modified to generate control PCR products in which the original probe sequences were replaced by a Drosophila probe sequence. Implementation of the coamplified controls increased our specimen throughput in comparison with the parallel control amplifications used previously. Clinical laboratories using PCR for diagnosis of infectious diseases may find positive controls particularly helpful for increasing confidence that negative amplifications represent truly negative specimens.

Base Sequence↗
PubMed1990

Selection of deletion mutants by polymerase chain reaction.

Polymerase chain reaction (PCR) based DNA amplification has replaced many time-consuming protocols in molecular biology. Here we describe a simple strategy to quickly select deletion mutants based on PCR methodology which then can be confirmed by nucleotide sequencing. A forward PCR primer is designed in such a way to recognize only the wild type sequences in the amplification reaction and thus a negative selection identifies the deletion in the samples.

Base Sequence↗
PubMed1990

Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.

Polymerase chain reactions (PCRs) synthesize abundant amplification products. Contamination of new PCRs with trace amounts of these products, called carry-over contamination, yields false positive results. Carry-over contamination from some previous PCR can be a significant problem, due both to the abundance of PCR products, and to the ideal structure of the contaminant material for re-amplification. We report that carry-over contamination can be controlled by the following two steps: (i) incorporating dUTP in all PCR products (by substituting dUPT for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primers; and (ii) treating all subsequent fully preassembled starting reactions with uracil DNA glycosylase (UDG), followed by thermal inactivation of UDG. UDG cleaves the uracil base from the phosphodiester backbone of uracil-containing DNA, but has no effect on natural (i.e., thymine-containing) DNA. The resulting apyrimidinic sites block replication by DNA polymerases, and are very labile to acid/base hydrolysis. Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.

DNA Glycosylases↗
PubMedDate not supplied

Miniemulsion droplets as single molecule nanoreactors for polymerase chain reaction.

Polymerase chain reaction (PCR) was successfully carried out inside stable and narrowly distributed water-in-oil nanodroplets with a size of 100-300 nm in diameter. The droplets were obtained by the miniemulsion process. Each aqueous droplet serves as a single nanoreactor for the PCR. It was found that the size of the droplets highly depends on the sonication parameters (i.e., time and amplitude) and that these parameters have a great influence on the final concentration of the PCR product. The parameters were chosen that way that conditions for single molecule chemistry were obtained, since the 3D-space is compartimentalized in small nanoreactors in each of which the same reaction takes place in a highly parallel fashion on every single DNA molecule.

Bioreactors↗
PubMedDate not supplied

[Study of transcription of the human cytochrome P450IID6 gene by polymerase chain reaction].

Polymerase chain reaction was used to study the expression of the drug metabolism gene. Primers complementary to the 2070-2090 and 2912-2930 sites within exons 4 and 6 of the gene CYP2D6 were synthesized. The amplification of the cDNA from total human liver mRNA was achieved. The length of the fragment obtained (238 bp) was in accordance with the distance between the primers binding sites in cDNA. The amplification of the DNA from the same source led to the longer fragment due to the presence of introns. The total RNA from the blood cells of the extensive metabolizers was shown to contain the mRNA transcribed from the CYP2D6 gene. The Taq polymerase reaction in the presence of cDNA derived from a poor metabolizer did not lead to the synthesis of the 238 bp fragment.

Base Sequence↗
PubMed1995

Immunoglobulin gene analysis of cutaneous pseudolymphoma by polymerase chain reaction.

Polymerase chain reaction (PCR) was used to amplify the V-D-J region of the immunoglobulin heavy chain gene from DNA extracted from the formalin fixed, paraffin embedded skin of 3 cases of pseudolymphoma. The products were electrophoresed and observed under ultraviolet light after ethidium bromide staining. Specimens of two cases showed smears of polyclonal amplified DNA. The specimen of one case (Case 3), however, showed a single band with a smear. The presence of monoclonality in B lymphocyte populations may suggest the possibility of low grade malignancy of pseudolymphoma or transformation to malignant lymphoma in the future.

Adult↗
PubMed1993

DNA and RNA amplification in urology: the polymerase chain reaction.

Polymerase chain reaction (PCR) technology is a promising molecular approach for diagnosis of genitourinary tract infections, inherited disorders, tumor-associated genetic alterations and tumor markers. Because it is based on exponential amplification, the technology is capable of ultimate theoretical sensitivity. Although not entirely free of technical limitations, PCR holds advantages over other diagnostic tests, including exquisite sensitivity, specificity, interchangeability and speed.

Base Sequence↗
PubMed1992

[DNA analysis by polymerase chain reaction].

Polymerase chain reaction (PCR) is a technique to amplify only a specific segment of DNA without using a plasmid or a phage vector. It is a powerful tool for genetic analysis of various diseases including inherited and viral diseases, and is now being applied to clinical diagnosis. Here, presented are several methods using PCR mainly for diagnosis of hemoglobinopathy which we have been engaged in. Some other diseases are also included.

Anemia, Sickle Cell↗
PubMed2000

[Detection of toxin-producing pathogenic bacterial strains by polymerase chain reaction].

Polymerase chain reaction (PCR) was used for detection of pathogenic Clostridium botulinum, Clostridium perfringens, Clostridium difficile, and Escherichia coli. With this aim in view, primers to botulinic toxins types A, B, C1, D, E, F, and G, perfringens enterotoxin, difficile toxin, and types 1 and 2 Shigella-like toxins were chosen and synthesized. Optimal amplification conditions were selected for each pair of primers, with DNA and the respective agent as the reaction mixture matrices. PCR was highly specific and sensitive in all cases. Its sensitivity was 10-100 cells/sample. Among the tested C. botulinum, C. perfringens, C. difficile, and E. coli strains, specific amplification products of expected size were observed only in the strains containing the respective toxin genes. These findings recommend the use of these methods in clinical microbiology. Strains containing type 2 Shigella-like toxin gene were detected among E. coli strains isolated from patients with the hemolytic uremic syndrome, which for the first time indicates that the problem with E. coli epidemic strain O157 is valid for Russia. As a result of our studies, test systems for detection of types A, B, C, D, E, F, and G C. botulinum strains, C. perfringens and C. difficile, and E. coli O157 strains are now available.

Bacterial Toxins↗
PubMed1993

Typing of Aspergillus species and Aspergillus fumigatus isolates by interrepeat polymerase chain reaction.

Polymerase chain reaction amplification of repetitive DNA motifs allows discrimination of Aspergillus species. In a preliminary survey, the DNA fingerprints appear to be identical when isolates of Aspergillus fumigatus are compared. When a primer deduced from a prokaryotic repeat motif is used, Aspergillus fumigatus isolates originating from different patients or different anatomical locations can be typed individually.

Aspergillus↗
PubMed1992

Detection of gene deletion in patients of Duchenne muscular dystrophy/Becker muscular dystrophy using polymerase chain reaction.

Polymerase chain reaction (PCR) was used to study the presence of gene deletion (the most prominent type of mutations) in some families afflicted by Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD). The results clearly demonstrate deletion in the central part of the DMD gene in two of the three families studied. This information can be useful for genetic counselling with particular reference to prenatal diagnosis and carrier analysis.

Base Sequence↗