PCR法

PCR法自1985年公布后一下子就成为了一项基础技术。能够从极微量的DNA中进行扩增的PCR法，也开始向RNA研究以及定量分析方面发展。现在也被广泛用于诊断、动植物育种等实用过程中。此讲由宝生物制品开发中心主任向井博之执行董事讲解。[编者按]     

A Fast-and-Robust Profiler for Improving Polymerase Chain Reaction Diagnostics

Polymerase chain reaction (PCR) is an in vitro technology in molecular genetics that progressively amplifies minimal copies of short DNA sequences in a fast and inexpensive manner. However, PCR performance is sensitive to suboptimal processing conditions. Compromised PCR conditions lead to artifacts and bias that downgrade the discriminatory power and reproducibility of the results. Promising attempts to resolve the PCR performance optimization issue have been guided by quality improvement tactics adopted in the past for industrial trials. Thus, orthogonal arrays (OAs) have been employed to program quick-and-easy structured experiments. Profiling of influences facilitates the quantification of effects that may counteract the detectability of amplified DNA fragments. Nevertheless, the attractive feature of reducing greatly the amount of work and expenditures by planning trials with saturated-unreplicated OA schemes is known to be relinquished in the subsequent analysis phase. This is because of an inherent incompatibility of ordinary multi-factorial comparison techniques to convert small yet dense datasets. Treating unreplicated-saturated data with either the analysis of variance (ANOVA) or regression models destroys the information extraction process. Both of those mentioned approaches are rendered blind to error since the examined effects absorb all available degrees of freedom. Therefore, in lack of approximating an experimental uncertainty, any outcome interpretation is rendered subjective. We propose a profiling method that permits the non-linear maximization of amplicon resolution by eliminating the necessity for direct error estimation. Our approach is distribution-free, calibration-free, simulation-free and sparsity-free with well-known power properties. It is also user-friendly by promoting rudimentary analytics. Testing our method on published amplicon count data, we found that the preponderant effect is the concentration of MgCl₂ (p<0.05) followed by the primer content (p<0.1) whilst the effects due to either the content of the deoxynucleotide (dNTP) or DNA remained dormant (p>0.1). Comparison of the proposed method with other stochastic approaches is also discussed. Our technique is expected to have extensive applications in genetics and biotechnology where there is a demand for cheap, expedient, and robust information.     

Biosensors Using Hybridization Chain Reaction - Design and Signal Amplification Strategies of Hybridization Chain Reaction

Signal amplification strategies are essential for sensitive and efficient detection. Among the recent amplification strategies, the hybridization chain reaction has been intensively studied because it has advantages of reaction at constant temperature and detection at low cost without specialized equipment. In this review, we have discussed how to adjust experimental conditions of the hybridization chain reaction and attractive signal amplification techniques including colorimetric, fluorescence, and electrochemistry. As a result, many studies using the hybridization chain reaction have been successful in detecting sensitive signals by enhancing the signaling of the various targets. These exciting features of the hybridization chain reaction have the potential to be widely used in many areas such as in situ disease diagnosis, food, and environmental analysis. Therefore, various platforms developed by applying this technology are expected to play an essential role as an efficient biosensor in many fields in the near future.     

聚合酶链反应

PCR是80年代中期发展起来的体外核酸扩增技术.它具有特异、敏感、产率高、快速、简便、重复性好、易自动化等突出的优点;能在一个试管内将所要研究的目的基因或某一DNA片段于数小时内扩增至十万乃至百万倍,使肉眼能直接观察和判断;可从一根毛发、一滴血、甚至一个细胞中扩增出足量的DNA供分析研究和检测鉴定.过去几天几星期才能做到的事情,用PCR几小时便可完成.PCR技术是生物医学领域中的一项革命性创举和里程碑.     

用PCR法直接快速筛查重组阳性克隆

本研究目的是应用PCR法快速筛查插入有苯丙氨酸脱氨酶（PAL）cDNA重组阳性克隆,用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALc DNA3’端终止密码TAA处的引物,以灭菌吸头挑一单菌落加PCR体系扩增。结果：在筛查的3个克隆中,有2个阳性克隆,并且插入方向正确,经DNA序列测定得到进一步证实,结果表明,以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方向,不需提取质粒。     

硅-玻璃聚合酶链式反应微芯片对β-葡糖苷酸酶基因的扩增

聚合酶链式反应（PCR）微芯片是基于微机电系统（MEMS）制作,在微芯片上进行PCR反应,实现生物样品扩增的一项新技术.介绍了硅-玻璃PCR微芯片的设计和制作、微反应腔的清洗和表面处理、借助外置温度控制系统进行PCR扩增反应以及扩增产物在琼脂糖凝胶电泳下的检测分析,实现了对β-葡糖苷酸酶（GUS）基因的有效扩增,扩增时间由原来的90 min缩短到现在的37 min.     

定量PCR的荧光技术

荧光定量PCR是在普通PCR基础上,利用荧光技术对核酸进行绝对定量的一项新兴技术,其灵敏度高、特异性高、操作简便和定量准确,已被广泛应用于临床和科研中。为更好地发挥荧光定量PCR的优点,荧光技术领域的研发工作十分活跃。     

基于荧光定量PCR扩增反应的SNP测定法

建立一种利用荧光定量PCR扩增反应进行单核苷酸多态性(SNP)快速测定的方法.以人β肾上腺素受体2基因中的Arg16Gly为研究对象,利用荧光染料SYBRGreenⅠ标记定量PCR产物,通过PCR生长曲线和融解曲线分析结果进行SNP分型.为提高SNP测定的特异性,分别在野生型和突变型等位基因的特异性引物3′端倒数第3个碱基位置,引入了一个人为错配碱基,使引物的错误延伸率显著降低,大大提高了SNP分析的准确性.通过DNA测序验证荧光定量PCR对β肾上腺素受体2基因中Arg16Gly分型结果的准确率.实验结果表明,所建立的方法操作简便,结果准确,适合进行大规模样品的SNP检测工作.     

实时荧光定量PCR的研究进展及应用

实时定量PCR(real-time PCR)的应用范围非常广泛,包括mRNA表达的研究、DNA拷贝数的检测、单核苷酸多态性(SNPs)的测定等。由于传统的PCR技术不能准确定量,使其在实际应用方面受到很大限制,因此,对PCR产物进行准确定量,尤其是病毒性病原的动态监控,成为迫切需要。     

Carcinogenesis: Evolution of concepts

Cancer is considered as an unintended consequence of internal imperfection of multicellular organisms: Darwinian evolution “does not foresee the future and does not plan for it”, it is forced to handle only anything that it has at a given moment “at hand”, which makes inevitable compromises and restrictions. In this case, there are a number of founding dogmas including mutagenesis as the main driving force of carcinogenesis; the environment as the main source of mutagenic effects; tumor monoclonality; cancer cell multistage transformation as Darwinian process of successive mutation—selection cycles. Recent discoveries complicate, supplement, and sometimes transform into an opposite fixed concepts. As a result, a new “image” of carcinogenesis is formed as a biological phenomenon whose conservation is indicative of its evolutionary utility.     

Genetic Applications of an Inverse Polymerase Chain Reaction

A method is presented for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle. This procedure of inverse PCR (IPCR) has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements. In this paper we show the feasibility of IPCR by amplifying the sequences that flank an IS1 element in the genome of a natural isolate of Escherichia coli.     

A New Primer Pair for Detection of Chlamydia pneumoniae by Polymerase Chain Reaction

In order to improve the detection and identification of Chlamydia pneumoniae, new primers for polymerase chain reaction (PCR) were designed based on the DNA base sequence within the 53-kDa protein gene, which is specific for C. pneumoniae. The primers permitted the identification of 24 C. pneumoniae strains collected from different geographical locations, but no reaction was observed with C. trachomatis, C. psittaci nor C. pecorum. The primers were unable to amplify the DNA of bacteria commonly related to respiratory tract infections. The positive amplification was achieved with only 9 EBs/assay. Therefore, the new primers seem to be useful in the diagnosis of C. pneumoniae infections.     

A Chain Reaction of Adrenaline Autoxidation is a Model of Quinoid Oxidation of Catecholamines

Biophysics - This review is focused on literature data and our own research of the nontrivial quinoid pathway for the oxidation of adrenaline. All catecholamines can be oxidized similarly with...     

Multistep carcinogenesis: a chain reaction of aneuploidizations

Carcinogenesis is a multistep process in which new, parasitic and polymorphic cancer cells evolve from a single, normal diploid cell. This normal cell is converted to a prospective cancer cell, alias "initiated", either by a carcinogen or spontaneously. The initiated cell typically does not have a new distinctive phenotype yet, but evolves spontaneously--over months to decades--to a clinical cancer. The cells of a primary cancer also evolve spontaneously towards more and more malignant phenotypes. The outstanding genotype of initiated and cancer cells is aneuploidy, an abnormal balance of chromosomes, which increases and varies in proportion with malignancy. The driving force of the spontaneous evolution of initiated and cancerous cells to ever more abnormal phenotypes is said to be their "genetic instability". However, since neither the instability of cancer phenotypes nor the characteristically slow kinetics of carcinogenesis are compatible with gene mutation, we propose here that the driving force of carcinogenesis is the inherent instability of aneuploid karyotypes. Aneuploidy renders chromosome structure and segregation error-prone, because it unbalances mitosis proteins and the many teams of enzymes that synthesize and maintain chromosomes. Thus, carcinogenesis is initiated by a random aneuploidy, which is induced either by a carcinogen or spontaneously. The resulting karyotype instability sets off a chain reaction of aneuploidizations, which generate ever more abnormal and eventually cancer-specific combinations and rearrangements of chromosomes. According to this hypothesis the many abnormal phenotypes of cancer are generated by abnormal dosages of thousands of aneuploid, but un-mutated genes.