等温扩增技术及其结合CRISPR在微生物快速检测中的研究进展

等温扩增技术因其对仪器依赖性低、核酸扩增高效等优势,非常适合于快速检测,已在微生物快速检测领域得到了广泛应用。本文从核酸提取、等温扩增(以环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)和重组酶聚合酶扩增技术(Recombinase polymerase amplification,RPA)为例)和产物检测角度,就近年来核酸等温扩增技术的发展及其在病原微生物核酸快速检测领域的应用进行综述,并概述了核酸等温扩增技术与CRISPR(Clustered regularly interspaced short palindromic repeats)基因编辑技术相结合的最新研究成果,为这些新兴技术的研究和未来的发展提供新思路。     

Application of nucleic acid amplification in clinical microbiology

The use of nucleic acid amplification methods in routine clinical microbiology laboratories is becoming increasingly widespread. The theory of polymerase chain reaction is described, including discussion of suitable microbal targets, extraction of nucleic acid from clinical samples, choice of primers, optimization of the process, laboratory design, contamination, and other problems as well as quality control. Other nucleic acid amplification methods such as ligase chain reaction, self-sustained sequence replication, strand displacement amplification, and branched DNA signal amplification are described and the choice of technology is discussed.     

Induction of selective DNA amplification and morphological cell transformation in Syrian hamster embryo cells

DNA amplification is a frequently observed event in continuous cell lines and in tumors. It is likely that a common mechanism underlies the amplification of specific DNA sequences which confer drug resistance and genes which give a growth advantage to the tumor. To find a correlation between the induction of DNA amplification by chemicals and morphological cell transformation we treated Syrian hamster embryo (SHE) cells with diverse antineoplastic agents of different classes. Analysis of these agents seems to be important since they are potentially carcinogenic and resistance inducing. For the measurement of DNA amplification we established a new system using adeno-associated virus type 2 (AAV)-infected primary SHE cells as target cells and amplification of viral DNA as marker of DNA amplification. Simultaneously we determined morphological cell transformation in SHE cells. Our findings demonstrate that there is only a limited correlation between the induction of AAV DNA amplification and the morphological cell transformation in SHE cells. The newly established system of AAV DNA amplification appears to be a useful tool for the investigation of drug resistance in target cells of choice.     

RNA扩增的研究进展

RNA扩增是伴随着基因芯片技术的应用而发展起来的一项技术,但不仅仅应用于芯片杂交。本文对RNA扩增的技术方法进行了综述,重点介绍了mRNA的线性扩增、指数扩增以及两者相结合的扩增方法以及优缺点,对microRNA的扩增方法也作了介绍,并对将来的发展作了展望。     

Critical evaluation of methods used to determine amplification efficiency refutes the exponential character of real-time PCR

Background  

The challenge of determining amplification efficiency has long been a predominant aspect of implementing real-time qPCR, playing a critical role in the accuracy and reliability that can be achieved. Based upon analysis of amplification profile position, standard curves are currently the gold standard for amplification efficiency determination. However, in addition to being highly resource intensive, the efficacy of this approach is limited by the necessary assumption that all samples are amplified with the same efficiency as predicted by a standard curve. These limitations have driven efforts to develop methods for determining amplification efficiency by analyzing the fluorescence readings from individual amplification reactions. The most prominent approach is based on analysis of the "log-linear region", founded upon the presumption that amplification efficiency is constant within this region. Nevertheless, a recently developed sigmoidal model has provided new insights that challenge such historically held views, dictating that amplification efficiency is not only dynamic, but is linearly coupled to amplicon DNA quantity. Called "linear regression of efficiency" or LRE, this kinetic-based approach redefines amplification efficiency as the maximal efficiency (E_max) generated at the onset of thermocycling.     

Gene amplification in cancer

Gene amplification is a copy number increase of a restricted region of a chromosome arm. It is prevalent in some tumors and is associated with overexpression of the amplified gene(s). Amplified DNA can be organized as extrachromosomal elements, as repeated units at a single locus or scattered throughout the genome. Common chromosomal fragile sites, defects in DNA replication or telomere dysfunction might promote amplification. Some regions of amplification are complex, yet elements of the pattern are reproduced in different tumor types. A genetic basis for amplification is suggested by its relative frequency in some tumor subtypes, and its occurrence in "early" preneoplastic lesions. Clinically, amplification has prognostic and diagnostic usefulness, and is a mechanism of acquired drug resistance.     

TempliPhi,phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.     

The Drosophila ACE3 chorion element autonomously induces amplification.

The Drosophila chorion genes amplify in the follicle cells by repeated rounds of reinitiation of DNA replication. ACE3 (amplification control element from the third chromosome) has been identified by a series of deletion experiments as an important control element for amplification of the third-chromosome chorion cluster. Several elements that quantitatively enhance amplification also have been defined. We show that a single 440-bp ACE3 sequence is sufficient to regulate amplification with proper developmental specificity autonomously from other chorion DNA sequences and regulatory elements. Although ACE3 is sufficient for amplification, the levels of amplification are low even when ACE3 is present in multiple copies. When controlled solely by ACE3, amplification initiates either at ACE3 or within closely linked sequences. Amplification of an ACE3 transposon insertion produces a gradient of amplified DNA that extends into flanking sequences approximately the same distance as does the amplification gradient at the endogenous chorion locus. The profile and extent of the amplified gradient imply that the low levels of amplification observed are the result of limited rounds of initiation of DNA replication. Transposon inserts containing multiple copies of ACE3 in a tandem, head-to-tail array are maintained stably in the chromosome. However, mobilization of the P-element transposons containing ACE3 multimers results in deletions within the array at a high frequency.     

Gene amplification system based on double rolling-circle replication as a model for oncogene-type amplification

Gene amplification contributes to a variety of biological phenomena, including malignant progression and drug resistance. However, details of the molecular mechanisms remain to be determined. Here, we have developed a gene amplification system in yeast and mammalian cells that is based on double rolling-circle replication (DRCR). Cre-lox system is used to efficiently induce DRCR utilizing a recombinational process coupled with replication. This system shows distinctive features seen in amplification of oncogenes and drug-resistance genes: (i) intra- and extrachromosomal amplification, (ii) intensive chromosome rearrangement and (iii) scattered-type amplification resembling those seen in cancer cells. This system can serve as a model for amplification of oncogenes and drug-resistance genes, and improve amplification systems used for making pharmaceutical proteins in mammalian cells.     

Alternative Molecular Tests for Virological Diagnosis

Several nucleic acid amplification techniques (NAATs), particularly PCR and real-time PCR, are currently used in the routine clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. However, conventional PCR methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. Therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. A new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. The main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. In this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional NAATs.     

DNA amplification in genetic toxicology

Chemical compounds can cause amplification of specific DNA sequences. DNA amplification may result in an enhanced production of gene products which help cells to cope with the chemicals. This may lead to a resistance of the cells toward the agent. Additionally, initiation of transformation or progression of transformed cells to tumorigenicity may also involve DNA amplification. Therefore, it is of interest to study the potential of chemicals to induce DNA amplification. This report focuses on the investigation of a variety of chemicals in 2 systems with which the amplification of viral DNA is measured within cells in culture. One model system comprises the measurement of SV40 DNA content in an SV40-transformed Chinese hamster cell line following chemical treatment. Antitumor agents as well as genotoxic and non-genotoxic compounds were studied in this system as a first step to determine the DNA amplification-inducing potential of a variety of differently acting chemical compounds. Also, a novel assay based on adeno-associated virus infection of cells is described. This system may offer the possibility of studying DNA amplification in a variety of different target cells. For the future, the need is stressed to develop and analyze versatile systems to study amplification of specific target genes in untransformed cells and in tumor cells.     

PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known single-base changes.

Previous data suggested that PCR amplification of specific alleles (also known as allele-specific amplification and amplification refractory mutation system) is not a general method for rapidly and accurately detecting known single-base changes. Herein we present our experience with the use of PCR amplification of specific alleles to detect 69 polymorphic or mutant alleles. Our results indicate that with proper optimization, all alleles were reliably distinguished.     

Cadmium induces cell centrosome amplification via reactive oxygen species as well as endoplasmic reticulum stress pathway

There is evidence that cadmium can initiate carcinogenesis. However, the underlying mechanisms remain unknown. There is also evidence that moderate centrosome amplification can initiate tumorigenesis. The present study investigated whether cadmium could trigger cell centrosome amplification, and examined the underlying molecular mechanisms. We found that cadmium was able to cause cell centrosome amplification at the subtoxic concentrations, in a dose-dependent manner. It could cause centrosome amplification via the signaling of reactive oxygen species (ROS). Proteomic analysis revealed that cadmium caused differential expressions of three proteins, which included HSPA1A which is associated with endoplasmic reticulum (ER) stress. Western blot analysis confirmed that cadmium upregulated HSPA1A. Further analyses showed that cadmium upregulated Bip and decreased the phosphorylation of ASK1 as well as increased the phosphorylation of MKK7 and c-Jun N-terminal kinases (JNK). Knockdown of JNK2 using small interfering RNA inhibited the cadmium-induced centrosome amplification but not the level of ROS. N-acetylcysteine did not inhibit the cadmium-activated ER stress pathway. In conclusion, our results suggest that cadmium can induce cell centrosome amplification via ROS as well as ER stress through the Bip–TRAF2–ASK1–MKK7–JNK signaling route, in parallel. More studies are required to clarify whether centrosome amplification underlies cadmium-induced carcinogenesis.     

Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87^N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.