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At least 19 recordsLinked to original sources
PubMed1995

In situ amplification of measles virus RNA by the self-sustained sequence replication reaction.

BACKGROUND: The self-sustained sequence replication (3SR) reaction is an isothermal method for nucleic acid amplification that has several features that make it an attractive alternative to PCR. We have studied the feasibility of the in situ 3SR reaction in cells using a measles virus-infected cell line as a model. EXPERIMENTAL DESIGN: The study was carried out in four steps. First, using RNA extracted from a measles-infected Vero Green monkey kidney cell line, conditions for the in vitro amplification of a segment of the nucleocapsid portion of the RNA viral genome were optimized for 420- and 119-bp 3SR products, and the results were compared. Second, 3SR was performed on intact infected cells in suspension, and the amount of RNA product was compared with infected cells without 3SR. Then, the 3SR reaction was conducted on cytospin preparation slides, followed by in situ hybridization for detection of the amplification product. Finally, 3SR was carried out on sections of formalin-fixed, paraffin-fixed, paraffin-embedded cells, and the degree of amplification as detected by ISH was quantified and compared between infected cells with and without 3SR reaction. RESULTS: Specific amplification of measles was observed in each of these types of preparations with an 8.5-fold rate of amplification in paraffin sections of formalin-fixed cells (a mean of 272.5 +/- 65.3 grains/cell after 3SR amplification in comparison to 31.97 +/- 4.2 grains/cell without amplification). CONCLUSIONS: A significant amount of amplification of RNA is possible with in situ 3SR (IS-3SR) and, in combination with ISH, offers several advantages compared with in situ PCR (IS-PCR), such as ease of use, lack of conditions that lead to cell damage, and a specificity for RNA amplification. This is the first report of specific amplification of RNA within cells using the IS-3SR procedure, a technique that has a wide range of potential applications in pathology and molecular biology.

Animals↗
PubMed1990

Unique features of the self-sustained sequence replication (3SR) reaction in the in vitro amplification of nucleic acids.

The development of a transcription-based amplification system and its application to a retrospective analysis of HIV-1-infected clinical samples demonstrated the specificity and sensitivity of this in vitro amplification procedure. The TAS protocol has been modified to mimic the retroviral strategy of replication, resulting in a self-sustained sequence replication (3SR) amplification reaction which operates under isothermal conditions (37 degrees C). The ability to specifically amplify only RNA sequences in the presence of DNA genomic copies containing the same sequence and the rapid kinetics of the 3SR reaction distinguish it from the well-used PCR protocol.

DNA Replication↗
PubMed1992

Detection of human immunodeficiency virus type 1 RNA in plasma samples from high-risk pediatric patients by using the self-sustained sequence replication reaction.

There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.

Base Sequence↗
PubMed1991

Use of self-sustained sequence replication amplification reaction to analyze and detect mutations in zidovudine-resistant human immunodeficiency virus.

Mutations at amino acid positions 67, 70, 215, and 219 in the human immunodeficiency virus type 1 (HIV-1) pol gene correlate with the emergence of resistance to zidovudine (AZT). These four positions were monitored in viral RNA extracted from infected peripheral blood mononuclear cells (PBMC) and viral stocks obtained after coculture with uninfected lymphocytes. Genotype determinations were made using the self-sustained sequence replication (3SR) and differential bead-based sandwich hybridization (BBSH) assay. The hybridization results obtained by 3SR and BBSH analyses were verified by dideoxynucleotide sequencing of the 3SR products. Correlation of 3SR and BBSH with polymerase chain reaction and Southern hybridization analyses of the PBMC and corresponding viral isolates indicated that PBMC and corresponding HIV-1 isolates may differ in their genotypes at the monitored amino acid positions, variations from the wild-type nucleotide sequence may occur proximal to the codons being monitored, and viral isolates possessing the same genotypes at the four monitored amino acid positions showed a threefold variation in their ID50 measurements.

Amino Acid Sequence↗
PubMed1996

Comparison of self-sustained sequence-replication reaction systems.

The 3SR (self-sustained sequence-replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro. This method requires three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H. We have modified the original protocol by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H. Comparison of the incorporation kinetics between the conventional three-enzyme 3SR and our two-enzyme 3SR shows differences in the kinetic behaviour. Furthermore, by the new two-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three-enzyme 3SR. The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments.

Base Sequence↗
PubMed1994

Emergence of a replicating species from an in vitro RNA evolution reaction.

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Avian Myeloblastosis Virus↗
PubMed1990

Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.

Base Sequence↗
PubMed1995

Applications of DNA amplification techniques in veterinary diagnostics.

An overview of the principles of the polymerase chain reaction, ligase chain reaction, self-sustained sequence replication and Q beta replicase is given. The application of these methods for the diagnosis of veterinary infectious and hereditary diseases as well as for other diagnostic purposes is discussed and comprehensive tables of reported assays are provided. Specific areas where these DNA-based amplification methods provide substantial advantages over traditional approaches are also highlighted. With regard to PCR-based assays for the detection of viral pathogens, this article is an update of a previous review by Belák and Ballagi-Pordány (1993).

Animals↗
PubMedDate not supplied

Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.

In situ hybridization (ISH) has proven to be a very important molecular tool in research and diagnosis. However, its applicability can be limited by its restricted detection sensitivity. During the last few years, several strategies have been developed to improve the threshold levels for ISH detection by amplification of either target nucleic acid sequences prior to ISH or the detection signals after hybridization procedures. In this overview, we outline and analyze the principles, applications, and limitations of in situ polymerase chain reaction, in situ self-sustained sequence replication, primed in situ labeling (PRINS), and in situ transcription as examples of target amplification methods, and catalyzed reporter deposition using biotinylated tyramine as an approach to signal amplification in ISH. We also provide a detailed, 1-day protocol for non-radioactive oligonucleotide ISH including signal amplification, which is suitable for diagnostic purposes. Furthermore, future directions of ISH including combined strategies of target and signal amplification as well as automation are discussed.

Forecasting↗
PubMed1994

A single temperature amplification technique applied to the detection of citrus tristeza viral RNA in plant nucleic acid extracts.

A procedure for the successful detection of citrus tristeza virus (CTV) RNA in total crude nucleic acid extracts of infected citrus whole leaves and bark is described. The method requires the isolation and precipitation of total nucleic acids from either infected whole leaf or bark tissue. The CTV viral RNA is then specifically amplified using a single temperature RNA Self-Sustained Sequence Replication technique (3SR) performed at 42 degrees C for 60 minutes. The amplified negative-sense viral RNA product can subsequently be detected by fixing a portion of the reaction mixture onto a nylon membrane and hybridizing with positive sense tristeza specific DNA oligonucleotide probes. Central California isolates of CTV were readily detected by this method. Denatured viral specific dsRNA was also a suitable template for the specific detection of CTV.

Base Sequence↗
PubMed1993

Use of probes and amplification techniques for the diagnosis and prognosis of human immunodeficiency virus (HIV-1) infections.

The recent development of nucleic acid amplification methodologies has markedly improved our ability to detect very low levels of specific nucleic acids. Amplification techniques have been combined with product detection systems that are designed for high throughput and are automatable. These developments are drastically changing the face of infectious disease diagnostics and changing the character of prognostic indicators in certain diseases. The polymerase chain reaction (PCR) has been used extensively for diagnosis of human immunodeficiency virus (HIV-1) infections, and recent developments have indicated that quantitative reverse-transcriptase PCR for viral RNA has prognostic value. Self-sustained sequence replication amplification for detection of viral RNA appears comparable to plasma culture for diagnosis of pediatric infections. The ligase chain reaction is still in developmental stages, but holds promise for specific purposes.

DNA, Viral↗
PubMed1993

Nucleic acid amplification technologies.

The polymerase chain reaction, Q beta replicase methodology, the ligase chain reaction, the self-sustained sequence replication system, and the new strand displacement assay have continued to progress with the development of improved reagents and new applications. These advances in enzymatic nucleic acid amplification strategies continue to provide research and medical communities with an ever-improving arsenal of ways to amplify RNA and DNA.

Gene Amplification↗
PubMed1997

A molecular predator and its prey: coupled isothermal amplification of nucleic acids.

BACKGROUND: A novel approach to the study of in vitro evolution is provided by the investigation of continuous, functionally coupled, amplifying systems. To date, in vitro evolution experiments have focused on issues of mutation and selection. Our work contributes to the new field of in vitro molecular ecology studies in which detailed information about the relationship between sequence changes and molecular interactions is obtained. Predator-prey systems are interesting in this context both in terms of evolutionary limits and in terms of the potential kinetic properties of oscillation and spatial pattern formation. Such molecular predator-prey models can be extended to a further negative-interaction mode, viral-host molecular evolution. RESULTS: A simple, nonfunctional predator-prey system based on the self-sustained sequence replication reaction is proposed. Coupling within the system is achieved using the single-stranded DNA intermediate of one cycle, the prey cycle, as primer for the second one, the predator cycle. Hybridization by complementary base pairing is the second order reaction step underlying the predation. Single steps of the whole reaction system have been investigated by radiolabeling. Each isolated subsystem operates according to the proposed reaction scheme, and evidence for an efficient coupling of both subsystems according to the proposed mechanism was found. CONCLUSIONS: Simple, interacting model systems based on nucleic acids can be designed and constructed for the study of coevolution. The results of studies such as the one described here will provide a basis for the construction of coupled systems of ribozymes, from which point the engineering of catalytic units for applications in biotechnology is feasible.

Base Sequence↗
PubMed1993

Photochemical sterilization of 3SR reactions.

The self-sustained sequence replication (3SR) reaction is an extremely efficient method for amplifying target DNA and RNA sequences that may be present in minute quantities. A serious problem often encountered in its practice is carryover contamination from products of previous 3SR reactions. A postamplification treatment of 3SR reaction products with the photoactive agent 4'-aminomethyl-4,5-dimethylisopsoralen (IP-10) was investigated as an approach for preventing carryover contamination by 3SR amplicons. Initially, inhibition of the amplification reaction by high concentrations of the reagent was observed. This problem was circumvented by developing a gel-based delivery of IP-10, and the method was found to provide highly efficient sterilization (approximately 10(6)-fold) of 3SR amplicons. Evaluation of this strategy on a number of 3SR targets has indicated that the degree of sterilization is dependent on the length of the amplified region and on the concentration of IP-10. It appears that the sterilization effect is caused by covalent modification of the pyrimidine bases of RNA and DNA, which renders them unusable as templates for the 3SR reaction. Modification of a purified RNA transcript with IP-10 was shown to prevent effectively reverse transcription by avian myeloblastosis virus reverse transcriptase (AMV RT). Similarly, treatment of a T7 RNA polymerase promoter-containing DNA template with IP-10 eliminated full-length transcription by T7 RNA polymerase. This isopsoralen method may be used to sterilize multiple 3SR reactions in a clinical assay with a convenient UV irradiation step.

Base Sequence↗
PubMed1997

Cooperative amplification of templates by cross-hybridization (CATCH).

In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model systems for the investigation of fundamental issues in evolutionary optimization. In this work we present a coupled amplification system based on the self-sustained sequence replication (3SR), also known as nucleic acid sequence-based amplification (NASBA), which allows the experimental investigation of evolving molecular cooperation. The 3SR reaction is an isothermal method of nucleic acid amplification and an alternative to PCR. A target nucleic acid sequence can be amplified exponentially in vitro using two enzymes: reverse transcriptase (RT) and a DNA-dependent RNA polymerase (RNAP). A system has been constructed in which amplification of two molecular species is cooperatively coupled. These species are single-stranded (ss)DNA templates (D1 and D2) of lengths 58 and 68 nucleotides, respectively. Coupling occurs when D1 and D2 anneal to each other via a complementary region (DB and DB') situated at the 3' end of each template. RT elongates the hybridized templates producing a double-stranded (ds)DNA of 106 base pairs (bp). This double strand contains two promoters, which are situated on either side of, and directly adjacent to DB, and which are oriented towards each other. These promoters specify two RNA transcripts encompassing, respectively, the D1 and D2 portion of the dsDNA. After hybridization of two primers (P1 and P2) to the transcripts (R1 and R2) and reverse transcription, the ss templates D1 and D2 are regenerated. Amplification cycles of D1 and D2 are coupled cooperatively via the common dsDNA intermediate. Under optimized batch conditions the system shows the expected growth phases: exponential, linear and saturation phase. The enzymes of the 3SR cycle tend to misincorporate nucleotides and to produce abortive products. In future experiments, we intend to use the system for studies of evolutionary processes in spatially distributed systems where new strategies for optimization at the molecular level are possible.

DNA-Directed RNA Polymerases↗
PubMed1993

Homogeneous detection of nucleic acids by transient-state polarized fluorescence.

We describe a transient-state polarized fluorescence-based method for detecting nucleic acids. An active ester of the phthalocyanine dye La Jolla Blue was coupled to an oligonucleotide containing an amino group at its 5' end, and the conjugate was purified by HPLC chromatography. We monitored the hybridization characteristics of the conjugate with complementary oligonucleotides and RNA as targets by transient-state polarized fluorescence measurements. The method was comparable in sensitivity to isotopic and nonisotopic heterogeneous detection systems and was capable of detecting 1 fmol of a 382-base-long RNA transcript from human immunodeficiency virus type (HIV-1) generated in a self-sustained sequence replication (3SR) reaction.

Base Sequence↗