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Polymerase chain reaction (PCR) has impacted nearly every field in molecular biology, genetics and forensic science. Numerous applications of PCR have been described for basic scientific purposes, as well as for diagnosis of hereditary and infectious diseases. This is particularly true for real-time PCR (RT-PCR), which detects and quantifies product molecules within PCR reaction vessels. However, PCR is still considered a daunting task due to many variables in the reaction, temperature cycling and complicated quantification methods. Current RT-PCR specific probes (Molecular Beacons, TaqMan, Scorpions) require costly synthesis and considerable effort to achieve optimal sensitivity. Typically, a fluorophore-quencher pair is attached to the ends of a probe, which doesn’t fluoresce when free in solution. Upon probe hybridization to an amplicon, the fluorophore is separated from the quencher and a signal is released.Recently we have discovered that the free energy of DNA quadruplexes can be used to drive unfavorable (endergonic) reactions of nucleic acids (e.g., isothermal PCR). The key point of quadruplex-driven reactions is that some G-rich sequences are capable of forming quadruplexes with significantly more favorable thermodynamics than the corresponding DNA duplexes. The energy of quadruplex formation can be used to drive PCR at constant temperature or DNA signal amplification. In addition, fluorescent nucleotide analogs incorporated and fully quenched within the primers regain maximum emission upon quadruplex formation, allowing very simple and accurate detection of product DNA. Thermodynamic and fluorescent bases of quadruplex priming amplification will be discussed.Supported by Bill & Melinda Gates Foundation.

A dramatic improvement in the rapidity and accuracy of laboratory testing for tuberculosis is anticipated in the near future from the application of molecular biology techniques. The polymerase chain reaction and other nucleic acid amplification methodologies have the potential to detect, amplify, and identify very small quantities of Myocobacterium tuberculosis DNA directly in a clinical specimen, even on the same day it is collected. Within the past 3 years, a number of polymerase chain reaction-based assays for tuberculosis have emerged. The development and evaluations of the polymerase chain reaction assay based on the insertion sequence IS6110 are described. For practical application in the clinical setting, amplification assays require a simple, reliable sample preparation method; an internal positive control to monitor for inhibitors; a method for eliminating contamination with amplicon (a polymerase chain reaction product) to prevent false-positive results; and a simple, sensitive detection method. The DNA fingerprinting method that uses IS6110 probes provides a means of differentiating individual strains of M tuberculosis and is a powerful tool for epidemiologic studies. The method and its applications are described.

A method for co-amplification of multiple viral sequences of HIV-1 and HIV-2 by polymerase chain reaction was designed. The technique resulted in the specific detection of each type of virus and allowed the amplification of as few as two copies of target DNA. The amplification of multiple regions of the viral genome offers the advantage of detecting multiple target sequences, which may be essential for some viruses, such as HIV, that exhibit a high degree of variability in their gene sequences. In addition, the method permitted the detection of both virus types in the same reaction, allowing for greater utility in geographic areas where coinfections with both viruses occur and cross-reactivity in Western blots is observed. This method was applied successfully to the detection of viral DNA in clinical specimens.

To isolate a novel gene that contains an SH2 domain, we devised a rapid and nonradioactive cDNA library screening method using polymerase chain reaction (PCR). For PCR amplification, we designed degenerate oligonucleotide primers from the multialigned DNA sequences of SH2 domains. This method offers an inexpensive and efficient approach for the isolation of clones of interest from cDNA libraries.

The enzymatic amplification of DNA directed by very short oligonucleotides of arbitrary sequence produces complex DNA profiles useful for genome analysis and identity testing. Mini-hairpins harboring a "core" arbitrary sequence at the 3' terminus primed the amplification of a wide range of templates ranging from plasmid DNA to plant and animal genomes. Primer core regions of only 3 nucleotides produced complex fingerprints, but the hairpin sequence also influenced the amplification reaction. Oligonucleotide substitution with degenerate bases tailored the complexity of fingerprint patterns. Simulation studies of the amplification of plasmids pUC18 and pBR322 using primers with short arbitrary cores allowed assignment of amplification products to expected regions and revealed physical interaction between annealing sites during amplification of first-round products. Mini-hairpin primers can increase detection of polymorphic DNA, and be used to study subgenomic fragments like yeast artificial chromosomes (YACs), cloned DNA and PCR products.

The polymerase chain reaction (PCR) is one of the most efficient techniques for measuring the viral load of HIV-infected samples. Determination of the specificity of PCR products is usually based on Southern blotting and hybridization of the amplified DNA to radioactive oligonucleotide probes specific for sequences comprised between the PCR primers. The recent introduction of capillary electrophoresis (CE) for identification of HIV-1 and HTLV-I PCR products appears interesting in light of its reproducibility, sensitivity and because it is fast and suitable for detection of DNA/DNA and DNA/RNA hybrids. We demonstrate that specific hybridization of a HIV-1 oligonucleotide probe to single-stranded DNA obtained by unbalanced PCR is detectable by capillary electrophoresis. This enabled us the application of a one-step, non-radioactive protocol to demonstrate the specificity of amplification of HIV-1 genomic sequences by PCR. This procedure is simple, reproducible and is suggested as an integral part of automated diagnostic systems based on the use of laboratory work stations for DNA isolation, preparation of PCR reactions and analysis of PCR products.

Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such 'failures' may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization. To examine these effects in solution-phase PCR, beta-globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4 degrees C, room temperature and 37 degrees C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37 degrees C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37 degrees C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.(ABSTRACT TRUNCATED AT 250 WORDS)

The detection and identification of microorganisms is being carried out increasingly using DNA. Each organism has a unique DNA sequence which can be used to distinguish closely related organisms. Using PCR amplification and sequencing of ribosomal RNA genes we have developed DNA probes for a number of pathogenic bacteria and fungi. The development of DNA assays based on PCR has resulted in new questions which must be addressed including process carry-over contamination and inhibition of the PCR amplification reaction once the problems associated with the implementation of DNA assays are ironed out.

Amplification of specific gene target sequences has become a routine molecular procedure in a variety of laboratories. When coupled with either a direct or indirect method of detecting amplified product, in situ amplification offers an extremely powerful investigative tool. We describe a protocol for a localized in situ amplification (LISA) reaction that includes tissue-culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections. Digoxigenin-11-dUTP was added to the amplification reaction and thus incorporated into the amplified products, providing a mechanism by which direct nonisotopic detection could be performed. To demonstrate the approach, LISA was performed on known positive Pneumocystis carinii rat lung tissues, with primers specific for the P. carinii rRNA gene sequence.

The polymerase chain reaction (PCR) has been extensively used in basic science research, and the clinical potential of PCR is only now beginning to be realized. The PCR is based on the fundamental DNA replication process that occurs in every living cell. PCR is essentially an in vitro adaptation of the in vivo DNA copying process. Because PCR is so efficient at amplifying even picogram quantities of DNA, contamination with even trace amounts of nucleic acids can lead to the generation of unwanted DNA sequences and false-positive test results. Despite this, there has been rapid growth in the use of PCR in biomedical research and clinical diagnostics. PCR is the most sensitive test for herpes simplex virus, varicella-zoster virus, and human papillomavirus infections. Other diagnostic uses, including tests for genetic diseases, cancers, and other infectious diseases, are evolving.

The polymerase chain reaction (PCR) has developed into one of the most promising methods for in vitro enzymatic amplification of DNA and has found widespread application in DNA cloning, sequencing and mutagenesis related studies. This innovative technique can selectively amplify a single target DNA molecule a billion-fold in a span of a few hours. Amplification of specific DNA sequences by PCR is useful in identification of sex, novel genes, pathogens and diseases. PCR has facilitated the establishment of evolutionary relationships among species and in revealing structural intricacies of single cells. In this article we review some of the major advances and applications of PCR, especially, its role in embryo sexing.

In 1985, Mullis discovered an exponentially increasing amplification system for nucleic acids, which became known as the polymerase chain reaction (PCR). This method permits the selective in vitro amplification of a particular DNA region by mimicking phenomena of in vivo DNA replication. Since then, many advances in PCR practice have been achieved, In this review, we will discuss the new techniques of in situ PCR, ligase chain reaction (LCR), transcription-based amplification system (TAS) and the isothermal replication system, self-sustained sequence replication (3SR). Many of the techniques described still remain largely research tools, however with further modifications they may become of use in routine diagnostic procedures in the next century.

The concept of whole genome amplification is something that has arisen in the past few years as the polymerase chain reaction (PCR) has been adapted to replicate regions of genomes that are of biological interest. The applications are many — forensic science, embryonic disease diagnosis, bioterrorism genome detection, ‘immortalization’ of clinical samples, microbial diversity, and genotyping. Several recent papers suggest that whole genomes can be replicated without bias or non-random distribution of the target, these findings open up a new avenue to molecular biology.

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

The widely used polymerase chain reaction (PCR) protocol requires several post-cycling steps to visualize amplicons, decelerating PCR sample processing and result calling. “One-stop PCR” was developed by including both the loading buffer and nontoxic staining dye within a single PCR tube, allowing direct loading and simultaneous staining of amplicons for visualization. The efficiency and sensitivity of one-stop PCR were comparable to commercially available PCR kits and it is compatible with downstream uses and is not mutagenic. One-stop PCR robustly minimizes the time and labor required for PCR reaction setup and product visualization. Key words : Polymerase chain reaction (PCR), post-cycling, compatible dye, Gelred.

Multiplex PCR allows the simultaneous amplification of up to dozens of target fragments in a single PCR. It is therefore a powerful tool to obtain many kilobases of continuous sequence from minute amounts of ancient DNA (aDNA), which usually must be amplified in multiple short and overlapping fragments. Because significantly less template is required compared to amplifying each fragment separately, multiplex PCR is particularly beneficial when the fossil material itself, or access to the fossil material, is limited. The recently refined two-step multiplex PCR protocol consists of a first-step reaction (the actual multiplex PCR) that then acts as the template for the second-step PCR. During the second step, nested primers are used in individual amplification reactions. Although the same set of primers can be used in both steps, using a nested set in the second step adds an additional level of selectivity and specificity, minimizing PCR artifacts. This is particularly important when complex mixtures of template DNA, such as aDNA extracts, are amplified.

PCR amplification of DNA is routine in modern molecular biology. However, the application of PCR to ancient DNA (aDNA) experiments often requires significant modification to standard protocols. The degraded nature of most aDNA fragments requires targeting shorter fragments, performing replicate amplifications, incorporating multiple negative controls, combating PCR inhibition, using specific DNA polymerases to deal with damaged bases, working in a separate aDNA facility, and modifying the PCR recipe to deal with damaged and low copy-number target DNA. In this chapter, we describe how and why these procedures are implemented, discuss aDNA-specific troubleshooting methodology, and suggest modifications to commercial cloning and sequencing procedures to reduce the expense of PCR product cloning.

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. There are three major steps involved in the PCR technique: denaturation, annealing, and extension. PCR is useful in the investigation and diagnosis of a growing number of diseases. Qualitative PCR can be used to detect not only human genes but also genes of bacteria and viruses. PCR is also used in forensics laboratories and is especially useful because only a tiny amount of original DNA is required. PCR can identify genes that have been implicated in the development of cancer. Molecular cloning has benefited from the emergence of PCR as a technique. The present paper is an attempt to review basics of PCR.

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR) and for optimizing this reaction for the sequence and primer set of interest. Important variables that can influence the outcome of PCR include the MgCl(2) concentration and the cycling temperatures. Additives that promote polymerase stability and processivity or increase hybridization stringency, and strategies that reduce nonspecific primer-template interactions, especially prior to the critical first cycle, can greatly improve sensitivity, specificity, and yield. This protocol is designed to optimize the reaction components and conditions in one or two stages. The first stage determines the optimal MgCl(2) concentration and screens several enhancing additives. To further improve specificity, sensitivity and yield, the second stage compares methods for optimizing initial specific hybridization to prevent polymerization of misprimed sequences prior to thermal cycling. For initial inhibition of polymerase activity, temperature (i.e., cooling reagents), physical separation ("hot start" method), and reversible antibody binding are compared.

Patient care providers face an enormous challenge in diagnosing vial respiratory diseases because of similar clinical manifestations, as well as insensitivity and/or slow conventional laboratory detection methods. Nucleic acid-targeted molecular assays are playing critical roles in rapid detection, screening, and identification of respiratory viral pathogens due to their high sensitivity and specificity, short test turnaround time, as well as automatic and high-throughput processing. User-developed and commercial molecular methods have gradually been developed and become available for detection and identification of common viral pathogens causing respiratory tract infections. Incorporated with cutting edge techniques, these methods can be used to detect one or more pathogens in a single reaction tube qualitatively and quantitatively. Although the molecular assays have been increasingly used in the clinical setting, laboratorians and clinicians should well know the limitations of these molecular assays to wisely choose the right tests and correctly interpret test results.

The next goals in the development of a synthetic biology that use artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and non-sequential non-standard nucleotides. This amplification must ensure that the non-standard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional non-standard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence “six-letter” GACTZP DNA. These are all not yet reported for any other synthetic genetic system.

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells.

Background Quantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. Results We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight) exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer). These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. Conclusions The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

Polymerase Chain Reaction (PCR) is a rapid procedure for in vitro enzymatic amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. Repetitive cycles involving template denaturation, primer annealing and the extension of the annealed primers by DNA polymerase, result in the exponential accumulation of a specific fragment whose termini are defined by 5′ end of the primers. The primer extension products synthesized in one cycle can serve as a template in the next. Hence the number of target DNA copies approximately doubles at every cycle. Since its inception, PCR has had an enormous impact in both basic and diagnostic aspects of molecular biology. Like the PCR itself, the number of applications has been accumulating exponentially. It is therefore recommended that relevant scientists and laboratories in developing countries like Nigeria should acquire this simple and relatively inexpensive, but rather robust technology.

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

The polymerase chain reaction (PCR) is a recently discovered technique of molecular biology that has revolutionized both basic science and clinical research laboratories. Its beautiful simplicity derives from its ability to geometrically amplify a single copy of a DNA sequence to millions of copies in a matter of hours. PCR thus allows both the molecular biologist to clone genes and the clinical pathologist to screen blood samples for human immunodeficiency virus, with rapidity and ease never before available. In this brief review we will discuss the basic technique of the PCR, its applications in the basic science and clinical areas, and further emphasize its current and potential roles in cardiovascular medicine.

INTRODUCTIONThe cornerstones of any genetic analysis study are the quality and quantity of the DNA samples. DNA is a precious limited resource, and in human disease studies the accessibility of sample DNA is often governed by the isolation method and the human source. Additionally, forensic analysis and archaeological research are generally infeasible without intact sample DNA. Therefore, mechanisms to preserve or enhance the quantity of the DNA stock are crucial to the success of these studies. Historically, to preserve and maintain DNA stocks, costly and labor-intensive Epstein-Barr-virus-transformed cell lines were produced. The creation of cell lines can be valuable for a number of reasons in addition to creating a renewable resource of DNA, but the cost and effort to create them, as well as the requirement of intact cells to begin with, limit the utility of this approach. More recently, whole-genome amplification (WGA), utilizing the unique property of the enzyme Φ29 DNA polymerase, has been used to generate robust high-fidelity copies of the genome. As described in this protocol, WGA using Φ29 DNA polymerase allows unbiased representation of the genome via multiple-strand displacement, followed by rolling-circle amplification on random primers.

With the advent of molecular-based methods to detect the presence of microbial pathogens, technologies such as the polymerase chain reaction (PCR) have revolutionized the field of food microbiology in ensuring that food products are pathogen free. In addition, these tests can also be applied to strain-specific identification, thus serving as an important tool for differentiating and identifying strains used in food production and as probiotics. The basis of these methods is the amplification of select sites in either DNA or RNA molecules such that the level of sensitivity is as low as one microbial cell or virus particle. Since the first days that PCR was rolled out and its potential as a powerful tool for microbial diagnostics was recognized, improvements in its chemistry and the machines that perform these reactions have been impressive. Real-time PCR, which enables concurrent amplification of nucleic acid target sites and the data readout indicating the status of the reaction, has reduced the time of analysis from 2–4 h to less than 1 h. Improvements in this technology have such potential that the detection and identification of all known microbial pathogens can be accomplished in one reaction. Many different types of molecular-based assays are described in this article.

Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Low efficiency is one of the limitations for conventional PCR amplification of random DNA sequence library in aptamer selection because of relative low products and high by-products formation efficiency. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.

In silico PCR analysis is a useful and efficient complementary method to ensure primer specificity for an extensive range of PCR applications from gene discovery, molecular diagnosis, and pathogen detection to forensic DNA typing. In silico PCR, SNPCheck, and Primer-BLAST are commonly used web-based in silico PCR tools. Their applications are discussed here in stepwise detail along with several examples, which aim to make it easier for the intended users to apply the tools. This virtual PCR method can assist in the selection of newly designed primers, identify potential mismatches in the primer binding sites due to known SNPs, and avoid the amplification of unwanted amplicons so that potential problems can be prevented before any "wet bench" experiment.

Polymerase chain reaction amplifications are finding increased applications in environmental microbiology. The development of sensitive and specific methods to detect amplified products is necessary especially when these amplifications are conducted in the presence of the environmental matrix. Gene probes specific to the npt11 locus were prepared by nick translation, 5’end labelling and by a PCR driven amplification. These probes were tested against a 300 bp PCR amplified segment of the npt11 region of the transposable element Tn5. The nick translated probe was the most sensitive, though not as specific as the other two types of probes. Sensitivity and specificity were found to be dependent on the hybridization format (Southern blots versus dot blots), the number of amplification cycles and on the purity of the target sequence.

The in-situ polymerase chain reaction (in-situ PCR) is a novel molecular technique that combines the extreme sensitivity of the PCR with the anatomical localization provided by in-situ hybridization. A number of groups have recently reported studies using in-situ PCR for the detection of specifically amplified single-copy nucleic acid sequences in single cell preparations or low copy DNA sequences in tissue sections. In this overview, we describe the principles of in-situ PCR, review the applications of this technique and discuss future aspects of in-situ PCR. We critically compare the different in-situ PCR protocols described in the literature. Emphasis is placed on the absolute requirement for controls to allow accurate interpretation of results and the possible problems and pitfalls of the in-situ PCR methods, including artefacts related to diffusion of PCR products and non-specific incorporation of labelled nucleotides into fragmented DNA undergoing repair. It is concluded that this technique will eventually play an important role in specialized diagnostic laboratories in the evaluation of viral diseases, haematological and other malignancies which have unique genetic markers.

Gene amplification is a common event in the progression of human cancers. The detection and quantitation of certain amplified oncogenes has been shown to have prognostic importance in certain human malignancies. A method is described that utilizes the principles of competitive PCR for quantitation of the c-myc gene copy number in relation to the copy number of a reference gene (tissue plasminogen activator [t-PA] gene) located on the same chromosome (8) as the c-myc gene. This ratio gives the true level of amplification of the c-myc gene, accounting for variables such as cell number, cell cycle phase, and chromosome 8 ploidy. The determination of gene amplification depends on the precise measurement of the ratio of target and reference genes. An important feature of this assay is that the competitive reference standards used for target gene c-myc and reference gene t-PA have been linked to form a hybrid. This simple modification guarantees that both reference gene and target gene assay tubes get identical amounts of the competitive template for each gene, thereby eliminating a significant source of error. This method has the same desirable attributes of standard PCR in that very small sample sizes are required and that results can easily be obtained in < 24 hr. In addition, this technique does not require the use of radioactivity or expensive DNA detection kits, and thus, may give it wider applicability for the study of human cancers.

Sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) permits rapid and sensitive detection of specific RNAs. However, the greatest advantage of RT-PCR, its remarkable sensitivity, has also limited its usefulness in quantitative applications, since the effects of minor variations in reaction conditions from sample to sample are greatly magnified during the amplification process. Several recently developed techniques circumvent this problem, allowing accurate quantitation of RNA using RT-PCR.

A new approach to simultaneous detection and typing of related agents by the multiplex polymerase chain reaction (PCR) is described. The reaction was been applied to human herpesviruses by nested amplification of fragments of the DNA polymerase genes. During the first amplification, primers were used as two equimolar mixtures of non-degenerate oligonucleotides, aligning the 3'-ends with selected consensus regions, and their 5'-ends with the non-related sequences of each herpesvirus to be amplified. The specific fragments obtained were the substrate for a second, multiplex reaction for which primers were designed to produce different-size fragments for each related virus. The results showed high specificity for the detection and typing of the human herpesviruses with known sequences and no amplification of human DNA, in spite of the presence of the same consensus regions within human DNA polymerase alpha. It is concluded that this new approach would be useful for the differential diagnosis of herpesviruses, as well as for other groups of agents with conserved regions in their genomes and causing similar syndromes.

The application of PCR to a wide variety of biological problems and molecular techniques has gained wide acceptance. RNA-PCR, a technique in which first-strand cDNA synthesis is followed by PCR amplification, has enabled detection and characterization of rare transcripts. One problem confronting the researcher involves specific amplification of transcribed sequences in the presence of small amounts of genomic DNA of identical sequence. We describe a novel technique, selective RNA amplification, which will specifically amplify RNA sequences in a background of homologous DNA. The method involves first-strand cDNA synthesis from a specific dUMP-containing oligonucleotide that contains unique user-defined 5' sequence (adapter sequence) not found in the message of interest. RNA template is degraded using RNase H, which is specific for RNA/DNA hybrids. This is followed by second-strand synthesis using a gene-specific primer (GSP). The original adapter primer is digested with uracil DNA glycosylase (UDG) to prevent its participation in subsequent amplification. PCR is then performed using the GSP and a second primer corresponding to the unique adapter sequence. In this paper, we apply this method to the amplification of RNA derived from human papilloma virus sequences. Using Southern analysis, we demonstrate specific amplification of 10(5) molecules of an in vitro-transcribed RNA. Denatured DNA of identical sequence and concentration was not amplified using the RNA-specific method. The method could eliminate the need for stringent purification of RNA and enables amplification of rare messages from RNA preparations containing homologous DNA of identical sequence and size.

The polymerase chain reaction (PCR) is a rapid and sensitive procedure for the in vitro amplification of specific segments of DNA. The ability to perform primer-directed amplification of specific sequences of DNA has had an effect on research similar to that of the discovery of restriction enzymes and Southern hybridization. Since the introduction of PCR in 1986, an ever increasing number of scientific applications have been reported, including the direct cloning, mutagenesis, sequencing and exact engineering of specific genes or gene sequences directly from genomic DNA or complementary DNA (cDNA). PCR is also being used increasingly for a variety of genetic and forensic analysis, as well as for several diagnostic procedures useful in screening for genetic diseases or disease agents. The success of PCR in generating and detecting specific DNA fragments has accelerated the use of molecular biology in many biological systems. The focus of this review is to offer examples of how PCR techniques may be used to study the molecular genetics, life cycles, ecology and phylogeny of filamentous fungi.

Summary.A simple and economic method for the detection and identification of human papillomaviruses (HPV) is described. The method has been developed with cloned HPV DNA and DNA from clinical samples. Genomic fragments were obtained from several different HPV types, including the ones most frequently encountered in the genital tract by polymerase chain reaction (PCR) amplification directed by degenerate general primers. The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. Different strategies are proposed, based on PCR and restriction analysis, and this approach to identification was compared with more classic methods such as Southern hybridisation.

The use of well-characterised, mapped DNA sequences was explored to prime polymerase chain reaction sequence amplification. Many of the markers that are been developed for use in genetic research are informative enough to be potentially useful in an industrial setting. Estimates of setup costs were also provided to utilise this technology and the throughput that could be expected of a laboratory facility managed by one worker

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.

Nucleic acid amplification methods, such as the polymerase chain reaction, are having a significant impact in the diagnosis of infectious diseases. Each of these methods uses an enzymatic replication of key regions of an organism's nucleic acid targets, such as the genome, rRNA, mRNA, or plasmid. Several methods and the diseases they are used to detect are described in the article. Techniques include PCR, AMPLICOR, AMPLIPREP, transcription-mediated amplification, thermophilic strand displacement amplification, nucleic acid sequence-based amplification, and the ligase chain reaction.

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator.

Proofreading DNA polymerase fusions offer several advantages for long-range PCR, including faster run times and higher fidelity compared with Taq-based enzymes. However, their use so far has been limited to amplification of small to mid-range targets. In this article, we present a modified protocol for using a DNA polymerase fusion to amplify genomic targets exceeding 20 kb in length. This procedure overcomes several limitations of Taq blends, which up until recently, were the only option for long-range PCR. With a proofreading DNA polymerase fusion, high-molecular-weight amplicon can be generated and analyzed in a single day, and a significant proportion is expected to be error-free.

Background ExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme. Results Because the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto cells can be transformed with a single plasmid or complex library, and then the expressed thermostable DNA polymerase can be used for PCR amplification. We demonstrate that ExCyto cells can be used to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours. Further, PCR amplification with ExCyto cells is comparable to amplification using commercial DNA polymerases. The ability to transform a bacterial strain and use the endogenously expressed protein for PCR has not previously been demonstrated. Conclusions ExCyto PCR reduces pipetting and greatly increases throughput for screening EST, genomic, BAC, cDNA, or SNP libraries. This technique is also more economical than traditional PCR and thus broadly useful to scientists who utilize analysis of cloned DNAs in their research.

Polymerase chain reaction (PCR) is a basic technique with wide applications in molecular biology. Despite the development of different methods with various modifications, the amplification of GC-rich DNA fragments is frequently troublesome due to the formation of complex secondary structure and poor denaturation. Given the fact that GC-rich genes are closely related to transcriptional regulation, transcriptional silencing, and disease progression, we developed a PCR method combining a stepwise procedure and a mixture of additives in the present work. Our study demonstrated that the PCR method could successfully amplify targeted DNA fragments up to 1.2 Kb with GC content as high as 83.5% from different species. Compared to all currently available methods, our work showed satisfactory, adaptable, fast and efficient (SAFE) results on the amplification of GC-rich targets, which provides a versatile and valuable tool for the diagnosis of genetic disorders and for the study of functions and regulations of various genes.

We have developed a highly sensitive single-molecule clonal amplification method called dual primer emulsion PCR (DPePCR) for next-generation DNA sequencing. The approach is similar in concept to standard emulsion PCR; however, in DPePCR both primers are attached to the beads, therefore following PCR amplification, both strands of the PCR products are attached to the beads. The ends of each strand can be freed for analysis by restriction digestion of the bridged PCR fragments, which allows efficient paired-end sequencing of fragment libraries.

Extraction of total DNA from a given habitat assumes significance in metagenomics, due to the requirement of inhibitor free and high quality metagenome in good quantity for applications in molecular biology. DNA extraction and its quality assessment for PCR applications from saline soils of Coastal Gujarat and Sambhar Soda Lake, Rajasthan in India is described in a comparative manner. The mechanical and soft lysis methods were simple and efficient for rapid isolation of PCR amplifiable total genomic DNA. The results are significant as only few extreme environments, particularly saline habitats are explored for their metagenomic potential.

Polymerase chain reaction (PCR) is a common method used to create copies of a specific target region of a DNA sequence and to produce large quantities of DNA. A few DNA molecules, which act as templates, are rapidly amplified by PCR into many billions of copies. PCR is a key technology in genome-based biological analysis, revolutionizing many life science fields such as medical diagnostics, food safety monitoring, and countermeasures against bioterrorism. Thus, many applications have been developed with the thermal cycling. For these PCR applications, one of the most important key factors is reduction in the data acquisition time. To reduce the acquisition time, it is necessary to decrease the temperature transition time between the high and low ends as much as possible. We have developed a novel rapid real-time PCR system based on rapid exchange of media maintained at different temperatures. This system consists of two thermal reservoirs and a reaction chamber for PCR observation. The temperature transition was achieved within 0.3 sec, and good thermal stability was achieved during thermal cycling with rapid exchange of circulating media. This system allows rigorous optimization of the temperatures required for each stage of the PCR processes. Resulting amplicons were confirmed by electrophoresis. Using the system, rapid DNA amplification was accomplished within 3.5 min, including initial heating and complete 50 PCR cycles. It clearly shows that the device could allow us faster temperature switching than the conventional conduction-based heating systems based on Peltier heating/cooling.