Polymerase chain reaction in situ: an appraisal of an emerging technique

Summary Polymerase chain reaction (PCR)in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCRin situ is, however, technically difficult, and amplification of the target DNA is only 30–300 fold. In this article we present an overview of PCRin situ techniques used to amplify both DNA and RNA targets (RT-PCRin situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks byTaq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.     

Polymerase chain reaction engineering

A mathematical model for polymerase chain reaction (PCR) is developed, taking into account the three steps in this process: melting of DNA; primer annealing; and DNA synthesis (polymerization). Activity and deactivation of the polymerase enzyme as a function of temperature is incorporated in the kinetic model to get a better understanding of the amplification of DNA. Computer simulation of the model is carried out to determine the effects of various parameters, such as the cycle number, initial DNA concentration (copynumber), initial enzyme concentration, extension time, temperature ramp, and enzyme deactivation on the DNA generation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 359-366, 1997.     

Polymerase chain reaction]

Principles of the polymerase chain reaction, its modifications and applications are discussed. Guidelines for performance of the polymerase chain reaction are briefly summarized.     

Polymerase chain reaction: a Markov process approach

A probabilistic approach to the kinetics of the polymerase chain reaction (PCR) is developed. The approach treats the primer extension step of PCR as a microscopic Markov process in which the molecules of deoxy-nucleoside triphosphate (dNTP) are bound to the 3' end of the primer strand one at a time. The binding probability rates are prescribed by combinatorial rules in accord with the microscopic chemical kinetics. As an example, a simple model based on this approach is proposed and analysed, and an exact solution for the probability distribution of lengths of synthesized DNA strands is found by analytical means. Using this solution, it is demonstrated that the model is able to reproduce the main features of PCR, such as extreme sensitivity to the variation of control parameters and the existence of an amplification plateau. A multidimensional optimization technique is used to find numerically the optimum values of control parameters which maximize the yield of the target sequence for a given PCR run while minimizing the overall run time.     

Polymerase chain reaction of nanoparticle-bound primers

Using one or two primers respectively bound to the surface of Au nanoparticles (AuNPs) or magnetic nanoparticles (MNPs), polymerase chain reaction (PCR) based on nanoparticles was systemically studied, agarose gel electrophoresis and atomic force microscopy (AFM) were respectively used to detect and observe the PCR product. The results obtained indicated that with either one or two primers respectively bound to the nanoparticle surface, PCR can proceed successfully under optimized condition and is subject to certain rules, consequently a symmetric PCR technique and an asymmetric PCR technique based on nanoparticles have been developed. A kind of nanostructured aggregates can be constructed by a symmetric PCR using two nanoparticle-bound primers.     

Polymerase chain reaction identification of Salmonella serotypes

Seventy-seven Salmonella isolates comprising 61 different serotypes were subjected to polymerase chain reaction (PCR) fingerprinting using two primersets. Primerset L1/G1, amplifying the spacer regions between the 16S and 23S rRNA genes, resulted in simple PCR fingerprints. However, in some cases PCR amplification of different Salmonella serotypes with primerset L1/G1 resulted in identical fingerprint profiles. Fingerprints obtained with the ERIC primerset, that matches the enterobacterial repetitive intergenic consensus sequence, were more complicated but were serotype-specific. Consequently, fingerprinting with the ERIC primerset is applicable for typing Salmonella up to the serotype level. Fingerprinting with the L1 and G1 primers requires an additional treatment of the amplification product for accurate typing of salmonellas. Phage typing is not possible with either primerset.     

FISH glossary: an overview of the fluorescence in situ hybridization technique

The introduction of FISH (fluorescence in situ hybridization) marked the beginning of a new era for the study of chromosome structure and function. As a combined molecular and cytological approach, the major advantage of this visually appealing technique resides in its unique ability to provide an intermediate degree of resolution between DNA analysis and chromosomal investigations while retaining information at the single-cell level. Used to support large-scale mapping and sequencing efforts related to the human genome project, FISH accuracy and versatility were subsequently capitalized on in biological and medical research, providing a wealth of diverse applications and FISH-based diagnostic assays. The diversification of the original FISH protocol into the impressive number of procedures available these days has been promoted throughout the years by a number of interconnected factors: the improvement in sensitivity, specificity and resolution, together with the advances in the fields of fluorescence microscopy and digital imaging, and the growing availability of genomic and bioinformatic resources. By assembling in a glossary format many of the "acronymed" FISH applications published so far, this review intends to celebrate the ability of FISH to re-invent itself and thus remain at the forefront of biomedical research.     

Polymerase chain reaction analysis of laboratory generated bioaerosols

The common methods for analyzing bioaerosols are based on maintaining organism viability and quantifying culturability which may result in the underestimation of microbial concentrations. The present study employed a well-developed technique that only requires cellular DNA to identify organisms. Polymerase chain reaction (PCR) was chosen to amplify specific DNA sequence from an organism, to detect and semi-quantify organisms. Suspensions ofFrancisella tularensis were aerosolized in a chamber, and air samples were collected using impingers. Samples were analyzed using limiting dilution PCR, and the results compared with those from a traditional plate counting. Results indicated that the limiting dilution PCR provides a new way to identify and quantify bioaerosols that does not rely on viability and culturability. Therefore, the method would provide a more reliable estimate of airborne bacterial concentrations compared to traditional plate counts.     

Polymerase chain reaction analysis of fragile X mutations

Summary The mutation that underlies the fragile X syndrome is presumed to be a large expansion in the number of CGG repeats within the gene FMR-1. The unusually GC-rich composition of the expanded region has impeded attempts to amplify it by the polymerase chain reaction (PCR). We have developed a PCR protocol that successfully amplifies the (CGG)_n region in normal, carrier and affected individuals. The PCR analysis of several large fragile X families is presented. The PCR results agree with those obtained by direct genomic Southern blot analyses. These favorable comparisons suggest that the PCR assay may be suitable for rapid testing for fragile X mutations and premutations and genetic screening of at-risk individuals.     

Polymerase chain reaction-based methods of DNA methylation analysis

DNA methylation is the main epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis. Hypermethylation of normally unmethylated CpG islands in the promoter regions often occurs in important tumor suppressor genes, DNA repair genes, and metastasis inhibitor genes. The changes of methylation status of various gene promoters seem to be a common feature of malignant cells and these changes can occur early in the progression process. Therefore detection of aberrant promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or detection of cancer recurrence. The purpose of this review is to provide a summary of the most commonly used techniques for the study of DNA methylation. Current scientific literature involving methylation detection methods was reviewed with an emphasis on polymerase chain reaction (PCR)-based detection methods. The current methodologies may be broadly classed into PCR-based methylation assays and non-PCR-based methylation assays. The problems and advantages of the different methods for detecting aberrant methylation are discussed. As the number of genes known to be hypermethylated in cancer is growing, the detection of aberrant promoter region methylation will be a promising approach for using DNA-based markers for the early detection of human cancers. Many techniques, especially PCR-based methylation assay techniques, make it practical to use these new methylation biomarkers in early cancer diagnosis.