Poliovirus RNA recombination: mechanistic studies in the absence of selection.

Direct and quantitative detection of recombinant RNA molecules by polymerase chain reaction (PCR) provides a novel method for studying recombination in RNA viruses without selection for viable progeny. The parental poliovirus strains used in this study contained polymorphic marker loci approximately 600 bases apart; both exhibited wild-type growth characteristics. We established conditions under which the amount of PCR product was linearly proportional to the amount of input template, and the reproducibility was high. Recombinant progeny were predominantly homologous and arose at frequencies up to 2 x 10(-3). Recombination events increased in frequency throughout replication, indicating that there is no viral RNA sequestration or inhibition of recombination late in infection as proposed in earlier genetic studies. Previous studies have demonstrated that poliovirus recombination occurs by a copy-choice mechanism in which the viral polymerase switches templates during negative-strand synthesis. Varying the relative amount of input parental virus markedly altered reciprocal recombination frequencies. This, in conjunction with the kinetics data, indicated that acceptor template concentration is a determinant of template switching frequency. Since positive strands greatly outnumber negative strands throughout poliovirus infection, this would explain the bias toward recombination during negative-strand synthesis.     

A method for counting PCR template molecules with application to next-generation sequencing

Amplification by polymerase chain reaction is often used in the preparation of template DNA molecules for next-generation sequencing. Amplification increases the number of available molecules for sequencing but changes the representation of the template molecules in the amplified product and introduces random errors. Such changes in representation hinder applications requiring accurate quantification of template molecules, such as allele calling or estimation of microbial diversity. We present a simple method to count the number of template molecules using degenerate bases and show that it improves genotyping accuracy and removes noise from PCR amplification. This method can be easily added to existing DNA library preparation techniques and can improve the accuracy of variant calling.     

The real-time polymerase chain reaction

The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.     

A closed tube format for amplification and detection of DNA based on energy transfer.

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.     

Seamless gene engineering using RNA- and DNA-overhang cloning

Here we describe two methods for generating DNA fragments with single-stranded overhangs, like those generated by the activity of many restriction enzymes, by simple methods that do not involve DNA digestion. The methods, RNA-overhang cloning (ROC) and DNA-overhang cloning (DOC), generate polymerase chain reaction (PCR) products composed of double-stranded (ds) DNA flanked by single-stranded (ss) RNA or DNA overhangs. The overhangs can be used to recombine DNA fragments at any sequence location, creating "perfect" chimeric genes composed of DNA fragments that have been joined without the insertion, deletion, or alteration of even a single base pair. The ROC method entails using PCR primers that contain regions of RNA sequence that cannot be copied by certain thermostable DNA polymerases. Using such a chimeric primer in PCR would yield a product with a 5' overhang identical to the sequence of the RNA component of the primer, which can be used for directional ligation of the amplified product to other preselected DNA molecules. This method provides complete control over both the length and sequence of the overhangs, and eliminates the need for restriction enzymes as tools for gene engineering.     

Direct sequencing of unpurified PCR-amplified DNA by semi-exponential cycle sequencing (SECS)

A simple technique for direct sequencing of PCR-amplified templates without purification of the PCR reaction product is presented. This method does not require an additional synthesis step after template amplification, and can generate sequence information form as little as 0.1 fmol of unpurified template.     

Event-specific qualitative and quantitative PCR methods for the detection of genetically modified rapeseed Oxy-235

Oxy-235 is an oxynil-tolerant genetically modified rapeseed approved for commercialized planting in Canada. The aim of this study was to establish event-specific qualitative and quantitative detection methods for Oxy-235. Both the 5'- and 3'-junction sequences spanning the plant DNA and the integrated gene construct of the Oxy-235 event were isolated, sequenced and analyzed. A 1298-bp deletion of the rapeseed genomic DNA that showed a high similarity to the mRNA sequence of Arabidopsis thaliana was found in the integration site of the insert DNA. Event-specific qualitative PCR methods were established, with one method producing a 105-bp product specific for the 5'-integration junction and the other method producing a 124-bp product specific for the 3'-junction. The absolute detection limits for the qualitative PCR were determined to be 100 initial template copies for the 5'-junction and ten for the 3'-junction. Quantitative methods were also developed that targeted both of the junction fragments. The limit of detection of the quantitative PCR analysis was ten initial template copies for either the 5'- or 3'-junction, while the limit of quantification was determined to be approximately 50 initial template copies. The real-time PCR systems so established were examined with two mixed rapeseed samples with known Oxy-235 contents and found to obtain the expected results.     

A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.

A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.     

基于PCR的质粒快速定点诱变方法建立与评价

基因突变对生命的进化具有重要意义,针对质粒的DNA定点诱变技术也是基因工程、蛋白质工程研究中的重要手段之一。为了提高质粒定点诱变的效率,本研究利用引物部分重叠的设计方案,使用Tm值相对固定的引物设计模式,将同一引物分为重叠区（Tm=50±2℃）和非重叠区（Tm=60±2℃）,并在严格控制模板用量（2 pg/kb）的基础上,通过20个PCR循环对目标质粒进行定点诱变扩增,随后取0.5 μL产物直接用于转化。在FastPfu Fly酶系中,利用此法构建了6个含碱基替换、缺失和插入的质粒,均获得成功,突变效率可达96%以上,阳性克隆获得数达70个以上。此外,利用4种不同PCR酶系对该法的适用性进行了评价,结果表明突变效率均可达93%以上,阳性克隆获得数均在10个以上。通过适当增加PCR模板用量（10 pg/kb）并使用纯化后的PCR产物进行转化,该法可适用于转化效率大于106（cfu/μg）的任意感受态细胞,对应的突变效率可大于91%,阳性克隆获得数大于20。根据本法的作用原理,该方案适合质粒中10～20 bp（因重叠区GC含量及碱基序列的不同而改变）以内的任意碱基替换和插入,以及任意长度的DNA片段缺失。且具有通用性强、耗时少、诱变成功率高、成本低、对感受态及转化效率无特殊要求等优点,适合各实验室的日常研究使用。     

Recombinant circle PCR and recombination PCR for site-specific mutagenesis without PCR product purification.

Two simple methods for site-specific mutagenesis are described and compared. In each method, the PCR is used in two separate amplifications to mutate the site of interest and to add ends to one PCR product that are homologous to the ends of the other PCR product. In the first method, the two products are combined, denatured and reannealed prior to transformation of E. coli in order to form recombinant circles in vitro, while in the second method, the two linear products are co-transfected directly into E. coli without prior manipulation, resulting in transformation of E. coli with the recombinant of interest by recombination in vivo. Each PCR amplification uses a plasmid template that has been linearized by restriction enzyme digestion outside the region to be amplified. This permits use of unpurified PCR products in these two protocols and generation of the mutant of interest with no other enzymatic manipulation in vitro apart from PCR amplification. In each protocol greater than or equal to 50% of the resulting clones contained the mutation of interest without detected errors.     

新定量PCR数据处理方法的理论探讨

日新月异的生命科学技术的发展及临床医学科学研究的需求,一般的PCR技术已远远不能满足工作的需要。PE公司在进行了大量的PCR动力学研究的基础上,发现了利用荧光标记探针在PCR循环过程中积累的荧光强度达到仪器捡出阈值时,系统的初始模板数量与循环次数之间有线性关系,据此建立了目前的PE 7700 、PE 5700仪器的定量PCR技术,开创了PCR技术的新局面。但是由于这一技术的误差较大,尚不能满足生命科学及临床医学科学研究的需求,因此需要继续研究新的定量PCR技术。PCR动力学数学模型是根据PCR 技术的原理提出的,能够准确描述PCR反应产物分子数量积累规律的动力学方程,给出了PCR产物数量或者荧光强度与初始模板数量及其他反应条件间的函数关系。利用这一关系,根据PCR反应已积累的产物数量,可以实现准确的定量PCR分析,得到初始模板数量达到定量PCR的目的。使用动力学数学模型做定量PCR分析,其结果的误差仅与使用的荧光强度数值的精确度相关。使用精确到6位数的荧光强度数据,模板数自100~1 000 000区间定量结果的准确性可达99%以上。本文根据模拟实验数据进行了初步的定量PCR分析,结果提示,目前的定量PCR仪器使用PCR动力学模型理论处理分析数据,定量分析的结果会比目前的CT值方法在准确性方面提高几十倍以上,可以满足各方面研究工作误差水平的需要。 Abstract:Today standard PCR can't satisfy the need of biotechnique development and clinical research any more.After numerous dynamic research,PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700.But the error of this technique is too great to satisfy the need of biotechnique development and clinical research.A better quantitative PCR technique is needed.The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system.This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions,and can reflect the accumulating rule of PCR product molecule accurately.Accurate quantitative PCR analysis can be made use this function relation.Accumulated PCR product quantity can be obtained from initial template number.Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used.For an example,when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1 000 000,the quantitative result accuracy will be more than 99%.The difference of result error is distinct using same condition,same instrument but different analysis method.Moreover,if the PCR quantitative analysis system is used to process data,it will get result 80 times of accuracy than using CT method.     

Sequencing PCR DNA amplified directly from a bacterial colony

We show that PCR product asymmetrically amplified directly from a bacterial colony can be sequenced to yield results as good as those obtained when purified template DNA is used for the PCR amplification step. With either template, greater than 300 nucleotides can be read from a typical sequencing reaction. Taq DNA polymerase was used for both the PCR amplification and sequencing reactions.     

Molecular colony diagnostics: detection and quantitation of viral nucleic acids by in-gel PCR

When PCR is carried out in a polyacrylamide gel, each target molecule forms a molecular colony that comprises many copies of the original template. By counting the number of colonies, one can directly determine the target titer, with 100% of the DNA molecules and approximately 15% of the RNA molecules being detected. Furthermore, because of the spatial separation of the products in the gel, no interference is observedfrom another simultaneously amplified target even if it is present at a 106 higher amount orfrom human nucleic acids that outweigh the target by up to a factor of 1,012, which is often true of clinical samples. All these features provide for an accurate and reliable assay of viruses even at very low amounts, that is, in cases most important to diagnostics.     

Universal TA cloning

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.     

应用RT-PCR检测基因的体外转录活性

体外转录分析已广泛应用于分子生物学领域．由于体外转录所产生的ＲＮＡ的量极少,需有灵敏的方法检测生成的ＲＮＡ．本研究发展了一种基于ＲＴ－ＰＣＲ技术鉴定体外转录产物的方法．将待研究基因的启动区与任何一段已知的含转录起始位点的编码序列相连,制备成体外转录的模板．体外转录后,用ＤＮａｓｅⅠ将ＤＮＡ模板完全降解;生成的ＲＮＡ经反转录合成ｃＤＮＡ;再以ｃＤＮＡ为模板,用相应的引物进行ＰＣＲ扩增,产物用琼脂糖凝胶电泳检测．该法灵敏度高,操作简便,无需同位素,且体外转录模板制备方便．还可结合其他技术,如Ｓｏｕｔｈｅｒｎ印迹分析,能获得更高的灵敏度．     

微量RNA的cDNA PCR文库的构建

使用PCR（polymerase chain reaction)技术,调制了mRNA的cDNA PCR文库,实验证明,cDNA PCR文库能使原cDNA的量放大数百倍,同时,使用人体K562培养细胞的总RNA,对cDNA PCR文库法和反转录中的β-Actin的cDNA量进行了比较,cDNA PCR文库法中的β-Actin的cDNA量大于高于反转录中的β-Actin的cDNA量,使用75pg的人体K562培养细胞的总RNA,调制成50ul的CDNA PCR文库,使用1ul的CDNA PCR文库进行了PCR反应时,可对文库中β-Actin的CDNA进行PCR检测,因此,CDNA PCR文库显示了良好的信息放大性能。     

A coalescent approach to the polymerase chain reaction.

A versatile algorithm is developed to model PCR on a computer. The method is based on a modification of the coalescent process and provides a general framework to analyse data from PCR. It allows for incorporation of the dynamics of the replication process as described in terms of the number of starting template molecules and cycle-dependent PCR efficiency. The simulation method generates, as a first step, the genealogy of a set of sequences sampled from a final PCR product. In a second step a mutation process is superimposed and the resulting data set is analysed. The efficiency of our algorithm enables us to get reliable approximations of various sample distributions. We demonstrate the relevance of our method with two applications: maximum likelihood estimation of the error rate in PCR and a test of homogeneity of the template.     

Detection of mitochondrial DNA deletion by a modified PCR method in a 60Co radiation-exposed patient

A new PCR based method was developed to detect deleted mitochondrial DNA (mtDNA). Peripheral blood cell DNA was obtained from a victim who was accidently exposed to a 60Co radiation source in 1990. Using the DNA as template, first PCR was performed to generate multiple products including true deletions and artifacts. The full length product was recovered and used as template of secondary PCR. The suspicious deletion product of mtDNA could be confirmed only if it was yielded by first PCR. Using either original primers or their nested primers, the suspicious deletion product was amplified and authenticated as a true deletion product. The template was recovered and determined to be a deletion by sequencing directly. The results show that a new mtDNA deletion, which spans 889 bp from nt 11688 to nt 12576, was detected in the peripheral blood cells of the victim. It indicates that this new PCR-based method was more efficient at detecting small populations of mtDNA deletion than other routine methods. MtDNA deletion was found in the victim, suggesting the relationship between the deletion and phenotypes of the disease.     

Accuracy in copy number calling by qPCR and PRT: a matter of DNA

The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods.     

Production of digoxigenin-labelled parvovirus DNA probe by PCR

A 560-bp digoxigenin(Dig)-labelled DNA-probe was produced by PCR using a 699-bp parvovirus DNA fragment as template with introduction of Dig-dTUP into the PCR reaction mixture.It was found to be very important to pay close attention to the amount of template employed, the number of cycles used, predenaturation of target DNA and optimization of the percentage of dTTP substituted by Dig-DUTP in the reaction mixture. The same 560-bp DNA fragment produced by PCR without the incorporation of Dig-DUTP in the reaction mixture, was subsequently labelled with Dig-dUTP by the random primed labelling method. Both of the Dig-labelled parvovirus DNA probes described above showed the same DNA detection level (about 1 pg), but production of the probe with Dig-DUTP incorporated in the PCR reaction mixture was much simpler.