The polymerase chain reaction

The polymerase chain reaction (PCR) is a novel technique that amplifies specific sequences with remarkable efficiency. Repeated cycles of denaturation, primer annealling and extension carried out with the heat stable enzyme, Taq polymerase, leads to exponential increases in the target DNA sequences. The technique has been widely applied in immunology to define HLA polymorphism, T-cell receptor and immunoglobulin diversity, pathogen detection, quantification of lymphokines and lymphoma or leukaemia detection. In addition, it has been very widely applied in all areas of molecular biology and human genetics. Because of its simplicity and sensitivity, PCR represents a major technical development for molecular biologists and will continue to have a great impact on molecular immunology. This review by John Bell describes the basic PCR technique and some variations on it and briefly explores their uses in molecular immunology.     

Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.     

Varicella-zoster virus-induced DNA polymerase.

Nuclear extracts of varicella-zoster virus (VZV)-infected human embryo lung (HEL) cells were found to contain DNA polymerase activity not present in uninfected HEL cells. This enzyme was designated the VZV-induced DNA polymerase. The VZV-induced polymerase was partially separated from the cellular alpha- and beta-polymerases by fractionation of the cells and by phosphocellulose chromatography. The separated enzymes were examined for the effect of added (NH4)2SO4, activity with synthetic templates, optimal pH, and the effect of phosphonoacetic acid. The VZV-induced DNA polymerase was distinct from cellular enzymes and had the properties of a typical herpesvirus-induced DNA polymerase.     

一种提高基因芯片检测中杂交信号强度的简单方法

目的 改进PCR产物和固定探针杂交效率。提高信噪比，以提高基因芯片检测的灵敏度。方法 以淋球菌隐蔽型质粒pJD1的基因芯片检测为例，在荧光素标记引物的PCR产物中加入不同浓度的Taq酶抑制剂EDTA及位于固定探针所在位点的两翼的荧光素标记探针，一起变性后再进行常规杂交、冲洗。结果 加入EDTA和荧光素标记探针使基因芯片检测的荧光强度能提高6倍以上。结论 加入EDTA和荧光素标记探针是一种提高基因芯片检测的效率的简单而有效的方法。     

Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase

The direct identification of enterotoxigenic Escherichia coli from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying the heat-labile toxin (LT) gene. Two synthetic primers, each of which was 20 bases in length, were used with the thermostable DNA polymerase from Thermus aquaticus to amplify the LT gene. The amplified PCR products were detected by either gel electrophoresis or hybridization to a 24-base synthetic oligonucleotide probe conjugated to alkaline phosphatase. The PCR method detected LT-positive bacteria but did not react with E. coli producing the heat-stable toxin, enteroinvasive E. coli, Salmonella typhi, Salmonella typhimurium, or Shigella dysenteriae. By the PCR method, a single bacterium could be detected following 30 cycles of amplification. The T. aquaticus DNA polymerase was inhibited by more than 10(3) organisms in the amplification reaction mixture. A group of 40 clinical specimens consisting of 16 LT bioassay-positive and 24 LT bioassay-negative stool specimens were tested by PCR for the presence of toxigenic E. coli. The total DNA from 100 microliters of stool specimen was extracted and partially purified with a commercially available ion-exchange column. All 16 of the bioassay-positive stool specimens were positive by PCR. In addition, one stool specimen which was bioassay negative for LT but positive for LT in a previous hybridization assay with a different LT probe was also positive by PCR. This may indicate that the LT gene is present but either is not expressed or is expressed below detectable levels. Amplification of specific DNA sequences by PCR provides a highly sensitive and specific tool for the detection of pathogenic microorganisms directly from clinical specimens without the need for prior isolation. This technique may find wide application in the detection of other organisms in addition to enterotoxigenic E.coli.     

Expansion of poxvirus RNA polymerase subunits sharing homology with corresponding subunits of RNA polymerase II

Poxvirus-encoded RNA polymerases were known previously to share extensive sequence homology in their two largest subunits with the corresponding subunits of cellular RNA polymerases and a modest alignment between the smallest poxvirus subunit and RBP10 of RNA polymerase II. The remaining subunits had no apparent cellular homologs. In this study, the HHpred program that combines amino acid sequence alignments with secondary structure predictions was used to search for homologs to the poxvirus RNA polymerase subunits. Significant matches of vaccinia RNA polymerase 22-, 19-, and 18-kDa subunits to RNA polymerase II subunits RPB5, 6, and 7, respectively, were identified. These results strengthen the concept that poxviral RNA polymerases likely evolved from cellular RNA polymerases.     

Monitoring hybridization during polymerase chain reaction

In analytical separation science, molecularly imprinted polymers have been applied in several analytical techniques, such as liquid chromatography, capillary electrochromatography and capillary electrophoresis, solid phase extraction, immunoassay, and as a selective sorbent in chemical sensors. A benefit of imprinted polymers is the possibility to prepare sorbents with selectivity pre-determined for a particular substance, or group of structural analogues. The application most close to a wider acceptance is probably that of solid phase extraction for clean-up of environmental and biological samples. The improved selectivity of imprinted polymers compared with conventional sorbents may lead to cleaner chromatographic traces in the subsequent analytical separation. Furthermore, the solid phase extraction application does not suffer from drawbacks generally associated with imprinted polymers in chromatography, such as peak broadening and tailing. Most liquid chromatographic studies have focused on using imprinted polymers as chiral stationary phases for enantiomer separations. Also, the use of imprinted polymers as selective sorbents in capillary electrochromatography has been presented. For this purpose, a protocol to prepare superporous, monolithic imprinted polymer-based capillary columns has been developed. Due to the high affinities and selectivities often achievable, imprinted polymers have been considered as alternative binding entities in biosensors and in immunoassay type protocols. Here, high stability, easy preparation and ability to be used for assay of both aqueous and organic solvent based samples are advantages of the polymers.     

Rhinovirus type 14 RNA polymerase complexes

Rhinovirus type 14 RNA-dependent RNA polymerase complexes were isolated from microsomal and soluble fraction of infected KB cells. Maximum activities were measured at at 6 and 7 hours post inoculation (p.i.) for microsomal and soluble polymerases, respectively. Both polymerase activities are considerably reduced by 8 to 9 hours, p.i., and interval in which the in vivo rate of synthesis of viral RNA is maximal. In vitro RNA products of RNA polymerases in both fractions consist of ribonuclease-sensitive and ribonuclease-resistant RNA of heterogeneous sizes. Detergent treatment of the microsomal RNA polymerase(s) did not affect the total amount of RNA synthesized, the proportion of ribonuclease-sensitive RNA synthesized or the size of the RNA products. The data suggest that RV14RNA polymerase complexes are intially associated with membranes but are then irreversibly released into the soluble phase of the cytoplasm; possible explanations for this phenomena are discussed.     

双链引物聚合酶链反应

目的　建立一种操作简单、不仅能够在反应开始之前发挥预防和降低非特异性扩增,而且能够在整个反应过程中都发挥这种作用的 Ｄ Ｎ Ａ 体外扩增技术。方法　人工合成脆性 Ｘ 智力低下１ 号基因（ Ｆ Ｍ Ｒ１）５′非翻译区序列特异单链引物和与其互补的引物,并将后者的３′末端进行修饰,使其失去延伸能力;最后将它们混合并退火成双链引物,再将双链引物加入 Ｐ Ｃ Ｒ 反应体系中进行热循环扩增。结果　在１５ 个标本的双链引物 Ｐ Ｃ Ｒ 反应中都得到了较使用常规 Ｐ Ｃ Ｒ 特异性更高的扩增产物。结论　双链引物 Ｐ Ｃ Ｒ 是一种特异性更好的 Ｄ Ｎ Ａ 体外扩增技术。     

DNA polymerase eta and chemotherapeutic agents

The discovery of human DNA polymerase eta (pol η) has a major impact on the fields of DNA replication/repair fields. Since the discovery of human pol η, a number of new DNA polymerases with the ability to bypass various DNA lesions have been discovered. Among these polymerases, pol η is the most extensively studied lesion bypass polymerase with a defined major biological function, that is, to replicate across the cyclobutane pyrimidine dimers introduced by UV irradiation. Cyclobutane pyrimidine dimer is a major DNA lesion that causes distortion of DNA structure and block the replicative DNA polymerases during DNA replication process. Genetic defects in the pol η gene, Rad30, results in a disease called xeroderma pigmentosum variant. This review focuses on the overall properties of pol η and the mechanism that involved in regulating its activity in cells. In addition, the role of pol η in the action of DNA-targeting anticancer compounds is also discussed.     

Cellular role of DNA polymerase I

Escherichia coli possesses three well-established DNA polymerases, I, II, and III. DNA polymerase I (Pol I) is the main repair polymerase in E. coli and also has a minor but important role in chromosomal replication. A major advantage of Pol I as an experimental system is its simplicity; unlike other replication enzymes, it is active as a single subunit. To a large extent, mutagenesis appears to be the result of (dis)functions of the DNA replication machinery. It is the purpose of this review to provide an integrated view of this relationship with particular emphasis on the role of Pol I in mutagenic events.     

The polymerase chain reaction in histopathology

Thanks to the advent of the polymerase chain reaction (PCR) molecular genetic study of histological samples is now a relatively straightforward task and the vast histopathology archives are now open to molecular analysis. In this review we outline technical aspects of PCR analysis of histological material and evaluate its application to the diagnosis and study of genetic, infectious and neoplastic disease. In addition, we describe a number of newly developed methods for the correlation of PCR analysis with histology, which will aid the understanding of the molecular basis of pathological processes.