Single-molecule PCR using water-in-oil emulsion

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.     

Rapid re-amplification of PCR products purified in low melting point agarose gels

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.     

Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR‐based single‐molecule sequencing approach

While standard DNA‐sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time‐consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large‐scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost‐effective manner. As a solution, we developed a simple PCR‐based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water‐in‐oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate.     

Identification and authentication of animal cell culture by polymerase chain reaction amplification and DNA sequencing

Polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis were used to identify the species origin of cell lines used in a cell culture facility where various cell lines of different species are routinely propagated. The aldolase gene family was selected for PCR amplification because the DNA sequences of this gene are highly conserved over a wide range of animals and humans. A total of 36 cell lines representing 13 different species were selected for this study. The DNA from each cell line was amplified, and PCR products were analyzed by agarose gel electrophoresis. The results showed unique profiles of amplified bands on agarose gels that allowed differentiation among non-closely related species. However, DNA amplification of closely related species, including rat and mouse or human and primate, resulted in similar and indistinguishable banding patterns that could be further differentiated by DNA sequence analysis. These results suggested that aldolase gene amplification coupled with DNA sequence analysis is a useful tool for identification of cell lines and has potential application for use in identification of interspecies cross-contamination.     

Highly sensitive and fast detection of Phoma tracheiphila by polymerase chain reaction

Summary A new method for the diagnosis of the plant pathogenic fungus Phoma tracheiphila has been developed. The method takes advantage of the enzymatic amplification of a specific 102 bp-long target sequence of fungal DNA by the polymerase chain reaction (PCR) using Thermus aquaticus DNA polymerase. The amplified DNA was characterized by agarose-gel electrophoresis, molecular hybridization using a synthetic oligonucleotide probe and direct sequencing. The application of the new method makes possible fast and direct detection of the pathogen in lignified plant tissues, a goal not previously achieved when a cloned probe and a dot-blot test were employed. In addition the PCR test can be used to advantage as a particularly simple and fast way of typing fungal isolates. This is achieved by submitting to DNA amplification crude homogenates of fungal mycelium and analysing the amplified DNA on an agarose mini-gel.Offprint requests to: F. Rollo     

三种提取柱状黄杆菌基因组DNA方法的比较

采用酚氯仿抽提法、CTAB法和SDS-蛋白酶K法分别对鱼类病原菌柱状黄杆菌提取基因组DNA。使用超微量紫外分光光度计和琼脂糖凝胶电泳检测所提取的基因组DNA的产量和质量,并用PCR扩增对DNA进行了评价。结果显示,3种方法均可提取到柱状黄杆菌的基因组DNA,并能有效扩增细菌16S rDNA序列,但CTAB法提取的DNA产量和质量最高,CTAB法可以作为柱状黄杆菌DNA提取以开展分子生物学研究的首选方法。     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Artificial duplicate reads in sequencing data of 454 Genome Sequencer FLX System

The 454 Genome Sequencer (GS) FLX System is one of the next-generation sequencing systems featured by long reads, high accuracy, and ultra-high throughput. Based on the mechanism of emulsion PCR, a unique DNA template would only generate a unique sequence read after being amplified and sequenced on GS FLX. However, biased amplification of DNA templates might occur in the process of emulsion PCR, which results in production of artificial duplicate reads. Under the condition that each DNA template is unique to another, 3.49%-18.14% of total reads in GS FLX-sequencing data were found to be artificial duplicate reads. These duplicate reads may lead to misunderstanding of sequencing data and special attention should be paid to the potential biases they introduced to the data.     

Premature termination of telomeric extension-PCR for detection of telomerase activity

In this article, we report a simple, rapid, and efficient method to detect telomerase activity: the premature termination of telomeric extension-PCR (PTEP). Similar to the telomeric repeat amplification protocol (TRAP), this method is based on PCR amplification following the in vitro telomerase reaction, while the in vitro telomerase reaction here is prematurely, rather than randomly, terminated. Apart from this, the telomeric extension products are used as initial primers, instead of as templates, to trigger the amplification with a specially constructed plasmid DNA as the template that cannot be directly amplified with the telomerase primer. The end product is a specific 159-bp DNA fragment that reflects telomerase activity. Because its product can be clearly identified with routine agarose gel electrophoresis and ethidium bromide staining, PTEP allows even lesser-equipped laboratories to easily detect telomerase activity.     

Efficient removal of PCR inhibitors using agarose-embedded DNA preparations.

The use of agarose blocks containing embedded DNA improves the PCR amplification from templates naturally contaminated with polysaccharides or humic acids, two powerful PCR inhibitors. Presumably, the difference in size between the DNA macromolecules and these contaminants allows their effective removal from the agarose blocks by diffusion during the washing steps, whereas genomic DNA remains trapped within them. In addition, agarose-embedded DNA can be directly used for PCR since low melting point agarose does not interfere with the reaction. This simple and inexpensive method is also convenient for genomic DNAs extracted by other procedures, and it is potentially useful for samples containing other kinds of soluble inhibitors, overcoming this important problem of current amplification techniques.     

Preparation of DNA suitable for PCR amplification from fresh or fixed single dinoflagellate cells

A method is described to prepare total DNA from single cells of dinoflagellates, which can be used for PCR amplification. As model organisms, we used a stock strain of Alexandrium catenella and cells of Dinophysis acuminata harvested from the Atlantic Ocean. Fresh grown cells or cells maintained in different preservatives were tested as sources for DNA preparation. The method used to prepare DNA combines physicochemical and enzymatic procedures on cells embedded in agarose plugs or beads. The agarose pieces containing the DNA were used to perform PCR amplification of a fragment of DNA containing a 5.8S rRNA gene and the flanking internal transcribed spacers (ITS1 and ITS2).     

Emulsion PCR: a high efficient way of PCR amplification of random DNA libraries in aptamer selection

Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Low efficiency is one of the limitations for conventional PCR amplification of random DNA sequence library in aptamer selection because of relative low products and high by-products formation efficiency. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.     

一种快速、经济提取外周凝血基因组DNA方法的建立

目的：建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法：摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用Ⅺ法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量。并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的Ⅺ法进行比较分析。结果：最佳的匀浆条件为：39000map,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论：与常规的外周凝血提取方法相比（蛋白酶K消化法）,本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。     

Non-isotopic PAGE analysis of amplified products from simple sequence repeat single-primer polymerase chain reaction

We describe modifications to two genetic typing procedures, simple sequence repeat (SSR)-anchored polymerase chain reaction (PCR) and single primer amplification of SSRs (SPARs), by combining polyacrylamide gel (PAGE) resolution of PCR amplified products with silver staining for detection. Turkey (Meleagris gallopavo) and chicken (Gallus domesticus) genomic DNA were used as templates in the PCR typing. The single primers used for PCR analyses were (CAC), (TCC), (GACT), (TGTC) and (TTTA). The PCR conditions have previously been described. As expected, the number of fragments detected by the analyses were higher than those previously described using agarose but lower than that reported by others from PAGE analyses and radioisotope detection. Unlike agarose gel analyses, all the primers amplified polymorphic products ranging from 22 percent (%) for (TGTC) to 44% for (TCC) (Table 1). The results suggest that the level of polymorphic DNA fragments from genetic typing by SPARs of SSRs could be increased, above that of agarose and ethidium bromide staining, by PAGE analyses followed by silver staining for detection. The level of DNA polymorphism detected was however lower than radioisotope detection, but the safety and ease of the modified method described in the current work may make it a preferable approach to both SPARs and SSR-anchored PCR for genetic mapping in eukaryotes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Amplification of bacterial genomic DNA by the polymerase chain reaction and direct sequencing after asymmetric amplification: application to the study of periplasmic permeases.

The polymerase chain reaction (PCR) has been used to amplify DNA fragments by using eucaryotic genomic DNA as a template. We show that bacterial genomic DNA can be used as a template for PCR amplification. We demonstrate that DNA fragments at least as large as 4,400 base pairs can be amplified with fidelity and that the amplified DNA can be used as a substrate for most operations involving DNA. We discuss problems inherent in the direct sequencing of the amplified product, one of the important exploitations of this methodology. We have solved the problems by developing an "asymmetric amplification" method in which one of the oligonucleotide primers is used in limiting amounts, thus allowing the accumulation of single-stranded copies of only one of the DNA strands. As an illustration of the use of PCR in bacteria, we have amplified, sequenced, and subcloned several DNA fragments carrying mutations in genes of the histidine permease operon. These mutations are part of a preliminary approach to studying protein-protein interactions in transport, and their nature is discussed.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60 degrees C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Start Codon Targeted (SCoT) Polymorphism: A Simple,Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.     

Uniform amplification of multiple DNAs by emulsion PCR

When several DNAs are amplified by PCR in one PCR tube, biased amplification is known to occur because amplification efficiency differs from one DNA to another. Therefore, we conducted PCR in the water in oil-emulsion (W/O emulsion) to examine whether the procedure allows the uniform amplification of several DNAs. In the amplification of a model library consisting of two clones, the emulsification of the PCR mixture successfully reduced the difference in its amplification efficiency to approximately one-seventh the value obtained without emulsification. Furthermore, we conducted repeated PCR to amplify a model library consisting of ten short hairpin RNA (shRNA) expression vectors as a model experiment for gene discovery using an shRNA expression library. Consequently, the emulsification of the PCR mixture successfully reduced PCR bias. Our results indicate that emulsion PCR is capable of uniformly amplifying libraries of shRNA, ribozyme, cDNA, and others, and is useful also for gene discovery using these libraries.     

Competitor internal standards for quantitative detection of mycoplasma DNA

Abstract Homologous internal controls were used as competitor DNA in the polymerase chain reaction for the quantitative detection of mycoplasma DNA. PCR primer sets were designed on the basis of the most conserved nucleotide sequences of the 16S rRNA gene of mycoplasma species. Amplification of this gene was examined in five different mycoplasma species: Mycoplasma orale, M. hyorhinus, M. synoviae, M. gallisepticum and M. pneumonias . To evaluate the primers, a number of different cell lines were assayed for the detection of mycoplasma infections. All positive cell lines showed a distinct product on agarose gels while uninfected cells showed no DNA amplification. Neither bacterial nor eukaryotic DNA produced any cross-reaction with the primers used, thus confirming their specificity. Internal control DNA to be used for quantitation was constructed by modifying the sizes of the wild-type amplified products and cloning them in plasmid vectors. These controls used the same primer binding sites as the wild-type and the amplified products were differentiated by a size difference. The detection limits for all the mycoplasma species by competitive quantitative PCR were estimated to range from 4 to 60 genome copies per assay as determined by ethidium bromide-stained agarose gels. These internal standards also serve as positive controls in PCR-based detection of mycoplasma DNA, and therefore accidental contamination of test samples with wild-type positive controls can be eliminated. The quantitative PCR method developed will be useful in monitoring the progression and significance of mycoplasma in the disease process.