Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

Frankia genus-specific characterization by polymerase chain reaction.

The polymerase chain reaction (PCR) is an in vitro procedure for primer-directed enzymatic amplification of specific template nucleic acid sequences. In order to determine whether a given actinomycete isolated from an actinorhiza (nodule) belongs to the genus Frankia or is a contaminant, we have developed a test based on the PCR. Primers complementary to sequences of two DNA regions corresponding to the nif genes (nifH and nifD) and the rRNA genes (16S and 23S) were specifically chosen to differentially amplify DNAs from Frankia strains but not those from other microorganisms. A series of positive and negative controls were set up by using universal or selective primers resulting in a discriminant amplification, which could be detected after agarose gel electrophoresis. In the nif region, degenerate oligonucleotide primers were used to amplify a target common to all the nitrogen-fixing microorganisms tested, while another set of primers amplified a target with a high specificity for Frankia strains. In the rRNA gene region, universal and specific primers were characterized and tested with DNAs from a wide range of microorganisms. The efficiency of this rapid and sensitive PCR assay was tested with an isolate obtained from Alnus nepalensis nodules, confirming results obtained by nodulation tests.     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.     

Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.     

Detection of beta-globin gene mutations by polymerase chain reaction.

The polymerase chain reaction and oligonucleotide probe hybridization technique were applied to the detection of two common mutations of the beta-globin gene found in Chinese, namely the 4-base pair deletion at the 41-42 codons and the C to T substitution at nucleotide 654 of IVS-2. The accuracy of the method was established using beta-thalassemia cases with known mutations or haplotypes of the restriction fragment length polymorphism (RFLP). A further 11 cases of thalassemia intermediate and thalassemia minor were then analysed with the same approach. Our results showed that 5 of the 11 cases carried the TCTT-deletion at codons 41-42. Our method is economical both in terms of materials and time needed and in an alternative to the use of the molecular RFLP approach in the prenatal diagnosis of beta-thalassemia.     

The gene for human complement component C9 mapped to chromosome 5 by polymerase chain reaction

The gene for human complement component C9 has been mapped to chromosome 5. This was achieved by using a novel application of the polymerase chain reaction to amplify specifically the human C9 gene on a background of rodent DNA in somatic cell hybrids. The assignment to chromosome 5 was confirmed by in situ hybridization to human metaphase chromosomes, giving a regional localization of 5p13.     

Improved retroviral vectors for gene transfer and expression

We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described.     

The polymerase chain reaction

This essay on the polymerase chain reaction is one of a series developed as part of FASEB's efforts to educate the general public, and the legislators whom it elects, about the benefits of fundamental biomedical research-particularly how investment in such research leads to scientific progress, improved health, and economic well-being.     

The polymerase chain reaction

The polymerase chain reaction (PCR) is a powerful new method for 'in vitro cloning'. It can selectively amplify a single molecule of template DNA several millionfold in a few hours and has made possible new approaches to problems in molecular genetics, evolutionary biology, and development.     

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.     

Detection and quantification of Listeria monocytogenes by 5'-nuclease polymerase chain reaction targeting the actA gene

AIMS: The aim of this study was to develop a 5'-nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes. METHODS AND RESULTS: Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 10(4) cfu ml(-1) after 35 cycles and 10(2) cfu ml(-1) after 45 cycles were achieved by PCR in both real-time and end-point fluorescence measurement modes. Linear calibration lines were obtained in the range from 10(2) to 10(9) cfu ml(-1) for three L. monocytogenes strains in real-time PCR with 45 cycles. CONCLUSIONS: The developed 5'-nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.     

Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction.

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.