Rapid and efficient site-directed mutagenesis by single-tube 'megaprimer' PCR method.

We describe a rapid and efficient megaprimer PCR procedure for site-directed mutagenesis that does not require any intermediate purification of DNA between the two rounds of PCR. This protocol is based on the design of forward and reverse flanking primers with significantly different melting temperatures ( T m). A megaprimer is synthesized in the first PCR reaction using a mutagenic primer, the low T m flanking primer and a low annealing temperature. The second PCR reaction is performed in the same tube as the first PCR and utilizes the high T m flanking primer, the megaprimer product of the first PCR and a high annealing temperature, which prevents priming by the low T m primer from the first PCR reaction. We have used this protocol with two different plasmids to produce cDNAs encoding seven distinct mutated proteins. We have observed an average mutagenesis efficiency of 82% in these experiments.     

PCR扩增试验的动力学数学模型

PCR技术已日趋成熟,但因为影响因素较多、反应过程比较复杂,直到目前PCR技术已创立近二十年,尚未能给出较好的描述PCR 反应的数学方法。我们根据它的基本原理提出了能够描述其反应过程的动力学方程：Wamp=［Ntarg×(1+P)n1+0.5×Cenz×U×P×Ceactiv×(n-n1)-Ntarg× (1+n×P)］×Cu×M,准确地描述了PCR反应的产物积累规律,建立了PCR反应的动力学数学模型。用动力学数学模型预测的PE 7700仪器的CT值与仪器的实际数值一致。动力学数学模型配合适当的监测设备可以构成自动化的PCR 定量仪器。PE 7700 仪器使用本动力学模型处理、分析数据,定量结果的准确性会更好。各实验室可根据各自的实验条件,由模型估算PCR产物数量,为PCR后产物继续处理提供较准确的数量信息。本模型阐明了PCR反应在多次循环后必然由指数扩增转变为线性扩增的分子基础,为定量PCR 提供了准确的计算方法。 Abstract:The PCR technique has been set up for nearly twenty years and is becoming more and more ripe.But because of the multiple influencing factors and complicated reaction procedures,no mathematical method that can describe the PCR reaction has been given.On the basis of its elementary principle,we suggested a kinetic equation to describe the reaction procedure,Wamp=［Ntarg×(1+P)n1+0.5×Cenz×U×P×Ceactive×(n-nl)-Ntarg×(1+n×P)］×Cu×M.This equation can describe correctly the accumulation rule of PCR product and thus build up the kinetic-mathematical model of PCR reaction.The predicted CT value of PE 7700 by the kinetic-mathematical model was in accordance with the real value detected by the machine.This kinetic-mathematical model accompanied by proper detecting equipment and computer could make an automatic PCR instrument,which would produce much better result.A laboratory can predict the amount of PCR product by this model and provide accurate information for further handling of PCR product according to its own condition.In this model,the molecular basis that PCR reaction is doomed to change from exponential amplification to linear amplification had been clarified.     

Estimation of the reaction efficiency in polymerase chain reaction

Polymerase chain reaction (PCR) is largely used in molecular biology for increasing the copy number of a specific DNA fragment. The succession of 20 replication cycles makes it possible to multiply the quantity of the fragment of interest by a factor of 1 million. The PCR technique has revolutionized genomics research. Several quantification methodologies are available to determine the DNA replication efficiency of the reaction which is the probability of replication of a DNA molecule at a replication cycle. We elaborate a quantification procedure based on the exponential phase and the early saturation phase of PCR. The reaction efficiency is supposed to be constant in the exponential phase, and decreasing in the saturation phase. We propose to model the PCR amplification process by a branching process which starts as a Galton-Watson branching process followed by a size-dependent process. Using this stochastic modelling and the conditional least-squares estimation method, we infer the reaction efficiency from a single PCR trajectory.     

On-line integration of PCR and cycle sequencing in capillaries: from human genomic DNA directly to called bases

A fully integrated system has been developed for genetic analysis based on direct sequencing of polymerase chain reaction (PCR) products. The instrument is based on a serially connected fused-silica capillary assembly. The technique involves the use of microreactors for small-volume PCR and for dye-terminator cycle-sequencing reaction, purification of the sequencing fragments, and separation of the purified DNA ladder. Four modifications to the normal PCR protocol allow the elimination of post-reaction purification. The use of capillaries as reaction vessels significantly reduced the required reaction time. True reduction in reagent cost is achieved by a novel sample preparation procedure where nanoliter volumes of templates and sequencing reaction reagent are mixed using a micro- syringe pump. The remaining stock solution of sequencing reaction reagent can be reused without contamination. The performance of the whole system is demonstrated by one-step sequencing of a specific 257-bp region in human chromosome DNA. Base calling for the smaller fragments is limited only by the resolving power of the gel. The system is simple, reliable and fast. The entire process from PCR to DNA separation is completed in ~4 h. Feasibilities for development of a fully automated sequencing system in the high-throughput format and future adaptation of this concept to a microchip are discussed.     

A general model of error-prone PCR

In this paper, we generalize a previously-described model of the error-prone polymerase chain reaction (PCR) reaction to conditions of arbitrarily variable amplification efficiency and initial population size. Generalisation of the model to these conditions improves the correspondence to observed and expected behaviours of PCR, and restricts the extent to which the model may explore sequence space for a prescribed set of parameters. Error-prone PCR in realistic reaction conditions is predicted to be less effective at generating grossly divergent sequences than the original model. The estimate of mutation rate per cycle by sampling sequences from an in vitro PCR experiment is correspondingly affected by the choice of model and parameters.     

Sex determination using the polymerase chain reaction

A practical class experiment on the PCR is described which has been used over several years as part of an undergraduate biochemistry and molecular biology course for science students. A major aim is to provide experience in the use of the polymerase chain reaction (PCR) and its interpretation. Students are given small coded DNA samples and use the PCR reaction to determine whether the sample is from a male or a female.     

免疫聚合酶链反应技术的建立及其对甲胎蛋白的检测

免疫ＰＣＲ是一种新的具高敏感性的抗原检测技术,它类似传统的ＥＬＩＳＡ法,若与抗体连接的酶用ＤＮＡ片段代替,则该ＤＮＡ片段可用于ＰＣＲ扩增。以链酶亲合素搭桥将生物素标记的抗体与ＤＮＡ相连建立了免疫ＰＣＲ技术,并将其用于检测甲胎蛋白（ＡＦＰ）,其敏感性比ＥＬＩＳＡ法高１０４。因此,免疫ＰＣＲ有可能作为一种具高敏感性的检测手段用于临床早期诊断     

Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction.

We describe a photochemical procedure for the sterilization of polynucleotides that are created by the Polymerase Chain Reaction (PCR). The procedure is based upon the blockage of Taq DNA polymerase when it encounters a photochemically modified base in a polynucleotide strand. We have discovered reagents that can be added to a PCR reaction mixture prior to amplification and tolerate the thermal cycles of PCR, are photoactivated after amplification, and damage a PCR strand in a manner that, should the damaged strand be carried over into a new reaction vessel, prevent it from functioning as a template for the PCR. These reagents, which are isopsoralen derivatives that form cyclobutane adducts with pyrimidine bases, are shown to stop Taq polymerase under conditions appropriate for the PCR process. We show that effective sterilization of PCR products requires the use of these reagents at concentrations that are tailored to the length and sequence of the PCR product and the level of amplification of the PCR protocol.     

Optimization of 6-carboxy-X-rhodamine concentration for real-time polymerase chain reaction using molecular beacon chemistry

The optimal 6-carboxy-X-rhodamine (ROX) concentration, which is used as a passive reference dye for real-time quantitative polymerase chain reaction (PCR) with molecular beacon chemistry, was determined with the Mx4000 Multiplex Quantitative PCR System. Additionally, the effects of changing ROX concentrations on PCR reproducibility, Ct values, and efficiency were investigated with this system by using the PCR data obtained from amplification of the Escherichia coli shiga toxin 2 (stx2) gene and the Campylobacter jejuni luxS gene. This study indicated that different ROX concentrations influence many aspects of the real-time PCR reaction. ROX concentration variation could have consequences in the analysis of quantitative data and may lead to erroneous results. This study further indicated that the optimal ROX concentration is 60 nmol/L for real-time PCR, using molecular beacon chemistry for PCR assay of luxS and stx2 genes.     

数字PCR技术在核酸标准物质研制中的应用

在标准物质研制领域,生物标准物质的研制逐渐成为了研究热点,同时基于核酸的检测技术的开发与应用又推动了核酸标准物质的进程。核酸标准物质需要高级别、精准的定值方法,数字PCR作为单分子定量技术得到了广泛的应用。数字PCR是一种测定核酸分子的绝对定量方法,如微滴式数字PCR是采用油包水形成的微滴作为反应室,将含有DNA模板的反应溶液分配到大量独立的反应室中进行扩增反应,再通过统计反应室中的阳性信号来定量DNA的拷贝数,从而达到精确定量核酸拷贝数的目的。综述了近年来关于数字PCR及其在核酸标准物质研究领域的最新应用进展,重点综述了其在转基因检测、医疗诊断等领域的应用进展,以期为核酸标准物质的研制提供参考。     

Apolipoprotein B(Arg3500----Gln) allele specific polymerase chain reaction: large-scale screening of pooled blood samples.

A two-step polymerase chain reaction (PCR) method for the rapid detection of the apolipoprotein B(Arg3500----Gln) mutation in a mixture of pooled blood samples is described. In the first step PCR, a short gene fragment surrounding codon 3500 is amplified. Subsequently the reaction product is subjected to a second amplification in which a mutation-specific primer is used. A PCR product is generated only if the mutant sequence is present in the DNA pool. Individuals carrying the mutation can then be identified by PCR with mutagenic primers and MspI restriction typing, essentially as described by Hansen et al. (J. Lipid Res. 1991. 32: 1229-1233).     

Validation and estimation of parameters for a general probabilistic model of the PCR process.

Earlier work rigorously derived a general probabilistic model for the PCR process that includes as a special case the Velikanov-Kapral model where all nucleotide reaction rates are the same. In this model, the probability of binding of deoxy-nucleoside triphosphate (dNTP) molecules with template strands is derived from the microscopic chemical kinetics. A recursive solution for the probability function of binding of dNTPs is developed for a single cycle and is used to calculate expected yield for a multicycle PCR. The model is able to reproduce important features of the PCR amplification process quantitatively.With a set of favorable reaction conditions, the amplification of the target sequence is fast enough to rapidly outnumber all side products. Furthermore, the final yield of the target sequence in a multicycle PCR run always approaches an asymptotic limit that is less than one. The amplification process itself is highly sensitive to initial concentrations and the reaction rates of addition to the template strand of each type of dNTP in the solution. This paper extends the earlier Saha model with a physics based model of the dependence of the reaction rates on temperature, and estimates parameters in this new model by nonlinear regression. The calibrated model is validated using RT-PCR data.     

实时荧光定量PCR技术及其应用

定量PCR的问世是继定性PCR(即常规PCR)后分子生物学方法学研究的一大飞跃,实现了对核酸信息量的分析比较,为疾病的诊断和治疗提供了更多、更有效的基因水平信息。本文介绍实时荧光定量PCR技术及其在兽医领域中的应用,包括近年来研究和应用较多的几种定量PCR技术。     

Evaluation of the analytical sensitivity of a polymerase chain reaction assay for the detection of chicken infectious anemia virus in avian vaccines

Chicken infectious anemia virus (CAV) is a ubiquitous pathogen of chickens causing significant disease in commercial flocks worldwide. During CAV outbreaks, the Center for Veterinary Biologics requires manufacturers of veterinary biologicals to test materials derived from infected flocks for extraneous CAV by polymerase chain reaction (PCR). The analytical sensitivity of a PCR assay for detection of CAV was determined and the applicability of a CAV DNA standard as a positive control for assay validity was evaluated. The analytical sensitivity of the CAV PCR assay was assessed to be 100 copies per reaction for the DNA standard and 1 × 10^1.9 TCID₅₀/reaction for infectious virus. Establishing the analytical sensitivity of this CAV PCR assay and the inclusion of internal and external positive controls for validity provide a basis for determining whether suspect materials are safe for use in the production of veterinary biologics.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.     

Chlamydomonas (Chlorophyceae) colony PCR

The ease and effectiveness of colony polymerase chain reaction (PCR) has allowed rapid amplification of DNA fragments and screening of large number of colonies of interest including transformants and mutants with genetic manipulations. Here, we evaluated colony PCR in Chlamydomonas. Individual colonies were treated with 10 mM ethylenediaminetetraacetic acid (EDTA) or Chelex-100 and the resulting clear cell lysate was used for PCR reaction. Either genomic DNA or plasmid DNA incorporated into the genome was equally amplified. We found that the Chelex method is superior to EDTA method in certain cases. This colony PCR technique will bypass the tedious process of isolating genomic DNA for PCR reaction and will make it possible for rapid amplification of genomic DNA fragments as well as rapid large-scale screening of transformants.     

Hot start of the polymerase chain reaction using DNA helicase]

A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.     

Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies

In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek''s microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: ● Set up reactions and thermal cycling conditions for a conventional PCR experiment ● Understand the function of various reaction components and their overall effect on a PCR experiment ● Design and optimize a PCR experiment for any DNA template ● Troubleshoot failed PCR experiments