利用MPprimer设计引物并优化扩增条件以提高多重PCR效率的实验研究

多重PCR技术广泛应用于多个研究领域,其中引物设计及扩增条件是提高多重PCR实验效率的关键因素.为探讨优化多重PCR实验的方法,以小鼠5个看家基因为研究对象,使用实验室新近开发的MPprimer程序设计多重PCR引物,并通过改变多种反应条件来优化多重PCR实验.结果表明,MPprimer程序能够设计出理想的多重PCR引物,并且通过对退火温度及延伸时间进行优化,可显著提高多重PCR实验效率,对于提高基因表达的规模化检测能力具有积极的促进作用.     

酶抗体应用实现热启动PCR

通过制备酶抗体，形成抗原抗体复合物，使得酶在较高的温度95℃,1 min,得以释放发挥聚合活性，实现热启动PCR.建立的热启动PCR体系灵敏度高，可以检测到1个拷贝的DNA分子，较普通PCR体系提高了3个数量级，并且具有较好的线性关系.这种通过形成抗原抗体复合物达到热启动PCR目的的方法的建立，具有着重要的意义，是提高PCR灵敏度和特异性的重要方法之一.     

数字PCR仪校准方法研究

数字PCR仪是核酸绝对定量的重要仪器,因此确保数字PCR仪检测结果的准确性十分重要。通过对国内市场上数字PCR仪的比较分析,剖析了数字PCR仪的性能指标,对数字PCR仪的校准方法进行了探讨,设定了拷贝数浓度相对示值误差、拷贝数浓度重复性、荧光通道一致性和反应单元个数重复性作为数字PCR仪整机校准的计量技术指标,采用具有溯源性的国家有证标准物质,对方法进行了试验验证。验证结果表明了校准思路和方法的可行性,该方法操作性强,能够满足仪器技术要求以及用户需求, 提高了数字PCR仪检测结果的准确性和可靠性,对进一步拓展和深化数字PCR 技术的应用具有积极的促进作用。     

荧光定量PCR应用常见问题

1荧光定量PCR是什么荧光定量PCR(fluorescent quantitative PCR,FQ- PCR)是在PCR定性技术基础上发展起来的定量PCR技术。FQ—PCR技术是一种在PCR反应体系中加入荧光基团,利用荧光信号积累实时监测整个PCR进程,最后通过标准曲线对未知模板进行定量分析的方法,实现了PCR从定性到定量的飞跃。FQ—PCR仪在普通PCR仪的基础上加上了荧光探头和计算机。     

PCR法对鸡卵黄中Coxiella burnetii菌的测定

[目的]通过用定量PCR加巢式PCR方法,提高了对Coxiella burnetii (C.b)CoMl基因的检出率;通过对鸡卵中病原微生物Coxiella burnetii的基因检测,明确鸡卵的食品安全性;并对明确Coxiella burnetii的流行病学有重要意义.[方法]提取鸡卵DNA,用定量PCR加巢式PCR方法检测上述基因,并对PCR产物进行测序分析,通过间接免疫荧光法观察鸡血白细胞中的微生物.[结果]用定量PCR加巢式PCR方法可检出4个以上的Coxiella burnetii Coml基因,用此方法可测出鸡卵中Coxiella burnetii Coml基因达104-106个,阳性率为5％-22％;对阳性鸡卵Coml基因PCR产物的测序结果显示有变异菌株的存在;免疫荧光法可见鸡卵中含有该微生物.[结论]由此认为鸡卵中存在病原微生物Coxiella burnetii,可能是Q热传染源.     

一种简便高效的改良降落PCR

降落(touchdown,TD)PCR通常涉及15个退火温度,程序设计复杂。报道了一种简便高效的改良降落PCR,只需5个降落退火温度,以杜氏盐藻(Dunaliellabardawil)基因组为模板,设计一对引物扩增胡萝卜素生物合成相关(carotenebiosynthesisrelated,cbr)基因的第3外显子。实验证实该方法程序简单,比标准降落PCR步骤简化70%,且产物的特异性及效率都有较大提高。     

PCR技术的新进展

PCR是用于在体外快速酶促扩增特定DNA片段的技术,本文对PCR技术的一些新方法进行了简要综述,包括易错PCR,DNA shuffilng,原位PCR,电子PCR,固相PCR和PCR芯片。     

一种简易的免疫PCR方法的建立

免疫PCR为一种高敏感度检测抗原的新技术,操作程序大多沿袭ELISA方法.用戊二醛作连接剂,将蛋白质高效率包被在普通PCR管内壁,使免疫及PCR反应用普通PCR仪得以在管中连贯地进行.实验结果表明,标准曲线的线性关系好,与ELISA方法比较敏感度高出约10⁵.这一改良法的建立,可望促进免疫PCR的普及应用.     

PCR技术研究进展

PCR（Polymerase chain reaction)在生命科学研究及相关诸多领域已经得到了广泛应用。本文对PCR技术的是新进展作一简要综述,包括：热循环仪的改进,引物的软件设计及网络设计,各种DNA聚合酶体系及其特性,多种PCR（Multiplex PCR),DNAshuffling,PCR芯片,固相PCR和电子PCR（Electronic PCR)的原理和应用。     

一种改进后的重叠延伸PCR法对被孢霉重组载体的构建

为了提高DNA大片段的拼接效率,通过引入逐次退火的PCR的方法,改良了传统的重叠延伸PCR方法。逐次退火PCR法,一方面延长了重叠区的PCR引物长度;另一方面把原来在1个循环中1个退火温度改成若干个,逐次降低退火温度,适用于Tm值相差比较大的引物;相邻的退火温度之间相差3-6℃。结果显示,通过此种方法成功拼接了ω3(2)和HCT两个大片段;PCR产物电泳条带单一,克隆测序证实序列完全正确,可以直接应用于后续试验。这种改进后的方法可以有效减少非特异性扩增,提高灵敏度,把这种方法称之为逐次退火重叠延伸PCR。     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

A rapid,efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5′ end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing.     

Factors affecting mitochondrial DNA quality from museum preserved Drosophila simulans

In this study we investigate how traditional killing methods and storage regimes affected mitochondrial DNA quality in Drosophila simulans. Here we define quality with three criteria: (1) size of extracted DNA, (2) extraction yield, and (3) ability to amplify from four target regions. Killing methods had a significant effect on extraction yield, but not on PCR success. Highest DNA yields were extracted from specimens exposed to cyanide, while the lowest were from specimens killed in 70% ethanol. Specimens stored for two years contained badly sheared DNA, which translated into a significant decrease in PCR success compared to freshly assayed specimens. The most dramatic decrease in PCR success occurred in the 1822 bp and 1332 bp amplicons, compared to the 959 bp and 291 bp fragments. Naphthalene did not affect any aspect of DNA quality; time of storage affected PCR success regardless of naphthalene environment. This study serves to further refine our understanding of DNA degradation.     

Introduction and PCR detection of Desulfomonile tiedjei in soil slurry microcosms

The aim of this work was to test the feasibility ofintroducing an anaerobic microbial reductivedechlorination activity into non sterile soil slurrymicrocosms by inoculation with the pure anaerobicbacterial strain Desulfomonile tiedjei, which iscapable of dechlorinating 3-chlorobenzoate tobenzoate. To show that the bacterium was establishedin the microcosms we followed the expression of thereductive dechlorination activity and a molecularprobe based on PCR amplification of the 16S rDNA genewas developed. However, the success of PCRamplification of the 16S rDNA gene depends on the DNAextraction and purification methodologies applied, asshown through the use of several protocols. In thisstudy we report a DNA extraction and purificationmethod which generates sufficient and very clean DNAsuitable for PCR amplification of the D. tiedjei16S rDNA gene. The threshold of detection was about5.10³ bacteria per gram of soil slurry.Introduction of D. tiedjei in soil slurrymicrocosms proved successful since 3-chlorobenzoatedechlorination activity was established with thisbacterium in microcosms normally devoid of thisdechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetateplus formate as cosubstrate, at their respectiveconcentrations of 5 and 6 mM, led to a totalbiotransformation of 2.5 mM of 3-chlorobenzoate within12 days. After complete 3-chlorobenzoatedechlorination, the 16S rDNA gene of this bacteriumwas specifically detected only in the inoculatedmicrocosms as shown by PCR amplification followed byrestriction mapping confirmation.     

Migratory route of Strongyloides venezuelensis in Lewis rats: Comparison of histological analyses and PCR

Strongyloides venezuelensis is a parasitic nematode that has been used as a model to study human and animal strongyloidiasis. In this study, we compared the sensitivity between traditional methodologies and PCR assay to characterize the dynamics of S. venezuelensis infection and its migration route in Lewis rats subcutaneously infected with 4000 L3. The dynamics of the infection was determined by counting the number of eggs and by detecting parasite deoxyribonucleic acid in faeces samples. Both techniques similarly detected the infection at day 6 after larvae inoculation. However, PCR performed with the genus primer showed higher sensitivity during the recovery phase. Histological analysis and PCR assay were then used to follow parasite tissue migration. S. venezuelensis migration route included the muscular fibers below the skin, the pulmonary alveoli and the small intestine vilosities. The sensitivity of these two techniques to detect parasite’s presence in these tissues was statistically similar.     

Expanding our understanding of immunoglobulin, T-cell antigen receptor, and novel immune-type receptor genes: a subset of the immunoglobulin gene superfamily

 The immunoglobulin superfamily (IgSF) is an extensively diversified multigene family whose members share a common structural feature, the Ig fold. Members of the Ig/T-cell antigen receptor (TCR) subset of the IgSF mediate antigen-specific recognition in adaptive immune responses. Antigen-binding receptors belonging to this subset are present in all species of jawed vertebrates. To explore whether there are additional structurally related but otherwise distinct members of this subset, we have developed a technique termed the short-primer polymerase chain reaction (PCR) that targets structurally conserved short motifs in the Ig fold. Large-scale sequencing efforts and recent advances in information biotechnology, including "electronic PCR," provide additional computational means to implement similarly directed searches within databases. The use of these approaches has led to the discoveries of Ig/TCR homologues in a variety of phylogenetically diverse organisms, a diversified family of novel immune-type receptor genes, as well as a novel human IgSF member. The potential of random sequencing efforts and virtual screening of databases is described in the context of two novel genes in bony fish. The various methodologies that are discussed and the examples shown provide means for further investigating, and/or elucidating novel, IgSF receptors as well as components of pathways that are involved in immune responses in both traditional and nontraditional model systems.     

PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food

AIMS: To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. METHODS AND RESULTS: Specificity of nuc targeted primers (Pri1-Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 10(3) CFU g(-1). The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93.3%). CONCLUSIONS: PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.     

Blastocystis: subtyping isolates using pyrosequencing technology

Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.     

简并PCR技术及其在基因克隆中的应用

本文简要介绍简并PCR技术,包括什么是简并引物,如何设计简并引物,进行简并PCR的反应条件,应用简并PCR获得全长基因的方法和简并PCR技术的应用范围,并对简并PCR技术的局限性及其新进展进行讨论。在此基础上,简述基因的克隆策略以及简并PCR技术在基因克隆中的应用。简并PCR技术是寻找和发现“新”基因或蛋白质家族新成员的一种非常有用的工具。 Abstract:Degenerate PCR is introduced in this paper,including what is degenerate PCR,how to design degenerate primers,how to optimize degenerate PCR parameters,how to applying degenerate PCR to obtain full-length gene and which fields can apply degenerate PCR.The limits and recent advances of degenerate PCR are also discussed.Based on this introduction,strategies of gene cloning and applications of degenerate PCR in gene cloning are summarized in brief.Degenerate PCR is a very useful tool for searching and discovering new genes and new members of a protein family.     

The Methodology Used to Measure Differential Gene Expression Affects the Outcome

Confirmation of gene expression by a second methodology is critical in order to detect false-positive findings associated with microarrays. However, the impact of methodology upon the measurement of gene expression has not been rigorously evaluated. In the current study, we compared differential gene expression between PC3 and PC3-M human prostate cancer cell lines using three separate methods: microarray, quantitative RT/PCR (qRT/PCR), and Northern blotting. The PC3 to PC3-M ratio of gene expression was determined for each of 24 different genes evaluated, by each of the three methods. Comparison of gene expression ratios between Northern and microarray, Northern and qRT/PCR, and microarray and qRT/PCR, gave correlation coefficients (r) of 0.72, 0.39, and 0.63, respectively. In each instance, one to two outlier genes were apparent. Their exclusion from analysis gave r values of 0.79, 0.72, and 0.83, respectively. These findings demonstrate that the assessment of differential gene expression is dependent upon the methodology used in each situation where outcome between different methodologies was compared, the presence of a relatively limited number of outlier genes precludes high overall correlation between the methods. Validation of gene expression by different methods should be performed whenever possible.