The Experts below are selected from a list of 4494 Experts worldwide ranked by ideXlab platform
Elektra Szewczuk - One of the best experts on this subject based on the ideXlab platform.
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Multiplexed tandem PCR: gene profiling from small amounts of RNA using SYBR Green detection
Nucleic acids research, 2005Co-Authors: Keith K. Stanley, Elektra SzewczukAbstract:Multiplexed tandem PCR (MT-PCR) is a process for highly multiplexed gene expression profiling. In the first step, multiple Primer pairs are added to the RNA to be analysed together with reverse transcriptase and Taq DNA polymerase. Following reverse transcription, the multiplexed amplicons are simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product is then diluted and analysed in multiple individual PCRs using Primers nested inside the Primers used for the multiplexed amplification. As the second PCR uses a template enriched in the amplicons of interest, the conditions can be optimized to significantly reduce ‘Primer Dimer’ formation allowing SYBR Green chemistry to be used for quantification. MT-PCR can be configured for as little as 10 pg RNA (equivalent to a single mammalian cell) and works well with RNA extracted from archival formalin-fixed paraffin-embedded sections. We illustrate MT-PCR with gene expression profiles of breast cancer cell lines.
Stephen Little - One of the best experts on this subject based on the ideXlab platform.
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A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping
Clinical chemistry, 1998Co-Authors: David Mark Whitcombe, Jannine Brownie, Jane Theaker, Clive R. Newton, Helen L. Gillard, Doug Mckechnie, Stephen LittleAbstract:We have developed a method whereby a single TaqMan™ probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of Primer-Dimer accumulation in PCR. The system uses a 5′-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.
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Adapting in situ polymerase chain reaction for genotyping of cells in suspension.
Diagnostic molecular pathology : the American journal of surgical pathology part B, 1998Co-Authors: Jeanine Brownie, Stephen Little, Lynn Paskins, Martin J. Schwarz, John H. BullAbstract:An approach is described for in situ polymerase chain reaction (ISPCR) based on cycling primed in situ synthesis (PRINS) conditions defined for alpha-satellite DNA. Using blood cell preparations subjected to limited fixation with paraformaldehyde, ISPCR cycling resulted in a gradual buildup of amplicon at the site of synthesis, as judged by the characteristic presence of paired nuclear spots corresponding to specific centromeres. Using longer cycling regimens, Primers for single copy genes also generated paired nuclear spots in a Primer-pair--specific manner. In this context, the amplification refractory mutation system (ARMS) was evaluated for in situ applications. In ARMS, allele-specific Primers are used in such a manner that PCR proceeds only when an exact 3' match between annealed Primer and template is recognized by DNA polymerase. Using normal and mutant Primers for the delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a model system, it was not possible to reliably differentiate between ARMS reactions by accumulation of direct labeled reaction product in cells, because of ARMS-independent nonspecific labeling. However, by DNA extraction and reamplification with ARMS Primers, it was shown that amplicon accumulates in cells in the expected Primer/template-dependent manner crucial to mutation detection by ARMS. It was also shown that nonspecific signal is due to Primer Dimer formation, especially in the absence of true template DNA. The impact of Primer Dimer formation in generating a false-positive signal is discussed. The method described here enables a cell population to be analyzed for a given point mutation.
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The elimination of Primer-Dimer accumulation in PCR.
Nucleic acids research, 1997Co-Authors: Jannine Brownie, Susan G. Shawcross, Jane Theaker, David Mark Whitcombe, Richard M. Ferrie, Clive R. Newton, Stephen LittleAbstract:We attempted to produce Primer-Dimers (PDs) from a variety of Primers with differing types and extents of complementarity. Where PDs were produced they were cloned and sequenced. We were unable to produce detectable PDs either with individual Primers alone or with similar sequence Primers even if they had 34 complementarity. These observations led to the hypothesis that a system could be developed whereby the accumulation of PDs in a PCR may be eliminated. We demonstrate a method for the general suppression of PD formation that uses a sequence of additional nucleotides (a Tail) at the 54 ends of amplimers. Tailed amplimers are present at low concentration and only participate during early cycles of PCR. In subsequent PCR cycles, amplification is achieved using a single Primer that has the same sequence as that of the Tail portion of the early cycle Primers, here we refer to this as a Tag. When products are small, as with PDs, there is a high local concentration of complementary sequences derived from the Tail. This favours the annealing of the complementary ends of a single strand produced by tailed Primer interactions and gives rise to ‘pan-handle’ structures. The formation of these outcompetes the annealing of further Tag Primers thereby preventing the accumulation of non-specific PD products. This aids the design of large multiplex reactions and provides a means of detecting specific amplicons directly in the reaction vessel by using an intercalating dye.
John M. Butler - One of the best experts on this subject based on the ideXlab platform.
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AutoDimer: a screening tool for Primer-Dimer and hairpin structures.
BioTechniques, 2004Co-Authors: Peter M. Vallone, John M. ButlerAbstract:The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applicat...
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multiplex pcr design strategy used for the simultaneous amplification of 10 y chromosome short tandem repeat str loci
Analytical and Bioanalytical Chemistry, 2003Co-Authors: Peter M. Vallone, Richard Schoske, Christian M Ruitberg, John M. ButlerAbstract:The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR Primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of Primers, or the use of chimeric Primers. The design of Primers, through the close examination of predicted DNA oligomer melting temperatures (T m) and Primer–Dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR Primer mixture constructed here revolves around varying the Primer concentrations rather than testing multiple Primer combinations. By solely adjusting Primer concentrations, a well-balanced set of amplicons should result if the Primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used.
Reza Parwaresch - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis of artifacts in the detection of telomerase activity and a modified Primer for a more robust ‘TRAP’ assay
Nucleic acids research, 1997Co-Authors: Guido Krupp, Karen Kühne, Sontka Tamm, Wolfram Klapper, Klaus Heidorn, Alexander Rott, Reza ParwareschAbstract:Human somatic cells have essentially no telomerase activity. Telomerase is linked to tumor genesis and is a valuable marker for malignant growth. Extreme paucity of the enzyme neccessitated development of a PCR-based assay, ‘telomeric repeat amplification protocol’ (TRAP). Unfortunately, this method is not without difficulties. Amplification products are not related to the size of the amplified telomerase products. Furthermore, false positive results can occur, and careful control of reaction conditions is crucial. We analyzed in detail the molecular basis of artifacts. Based on these data, reverse PCR Primer was changed and both problems in the TRAP assay were eliminated. Telomerase is a ribonucleoprotein with the function of a DNA polymerase, where a segment of the RNA component functions as internal template. 34-termini of linear chromosomes are the natural Primers for telomerase. It adds telomer hexamer repeats, with substantial pausing after the addition of each hexamer. In vertebrates, the repeat sequence is TTAGGG (1). Telomerase activity is a potentially very useful tumor marker. A sensitive and reliable assay is of high interest, but it is hampered by the extreme paucity of the enzyme. For in vitro assays, synthetic non-telomeric oligonucleotides can be used as Primers (2) and Kim et al. (3) developed the ‘telomeric repeat amplification protocol’ (TRAP), where telomerase products are amplified by a subsequent PCR (Fig. 1). Already in the presentation of the method, a peculiar artifact was described (3). Amplification of a well defined, synthetic telomerase product which contains four repeats (oligo TL) does not result in a single product of original size. Instead, a product ladder with 6 base pair steps extends up to the top of the gel. This is identical to the result observed in an authentic positive telomerase assay. Supposedly, this is caused by staggered annealing of CX Primer (3) (see Figs 1 and 2A). Although T:T mismatches in Primer CX reduced the artifact, an efficient suppression was not achieved. We had the following apprehension: highly variable amounts of the tentative ‘Primer Dimer’ were observed ( 3,4). If Primers CX and TS yield a real Primer Dimer, this is very similar to TL (Figs
Yongxi Zhao - One of the best experts on this subject based on the ideXlab platform.
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Single copy-sensitive electrochemical assay for circulating methylated DNA in clinical samples with ultrahigh specificity based on a sequential discrimination-amplification strategy.
Chemical science, 2017Co-Authors: Wang Xuyao, Feng Chen, Dexin Zhang, Chunhai Fan, Yue Zhao, Jing Wei, Lihua Wang, Shiping Song, Yongxi ZhaoAbstract:Tumor-related circulating methylated DNA represents only a small fraction of the total DNA in clinical samples (e.g. plasma), challenging the accurate analysis of specific DNA methylation patterns. Yet conventional assays based on the real-time quantitative methylation-specific PCR (qMSP) are generally limited in detection sensitivity and specificity due to its non-specific amplification interference including Primer Dimers and off-target amplification. Here we propose a single copy-sensitive electrochemical assay for circulating methylated DNA with ultrahigh specificity on the basis of a sequential discrimination–amplification strategy. Methylated DNA rather than unmethylated DNA in a bisulfite-modified sample is identified and amplified by the asymmetric MSP to generate abundant biotin-labeled single-stranded amplicons with reduced Primer–Dimer artifacts. Self-assembled tetrahedral DNA probes, which are readily decorated on an electrode surface as nanostructured probes with ordered orientation and well controlled spacing, enable the highly efficient hybridization of the specific single-stranded amplicons due to greatly increased target accessibility and significantly decreased noise. The interfacial hybridization event is quantitatively translated into electrochemical signals utilizing an enzymatic amplification. The proposed assay integrates dual sequence discrimination processes and cascade signal amplification processes, achieving the identification of as few as one methylated DNA molecule in the presence of a 1000-fold excess of unmethylated alleles. Furthermore, the excellent assay performance enables tumor related methylation detection in lung cancer patients with 200 microlitre plasma samples. The results are in good consistency with those of clinical diagnosis, whereas the conventional qMSP failed to detect the corresponding methylation pattern of these clinically confirmed positive patients in such trace amounts of samples.
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Zero-Background Helicase-Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts
Chembiochem : a European journal of chemical biology, 2016Co-Authors: Feng Chen, Dexin Zhang, Qing Zhang, Xiaolei Zuo, Chunhai Fan, Yongxi ZhaoAbstract:Primer-Dimer artifacts resulting from unintended template-independent Primer-Primer interactions often hinder the specific amplification of nucleic acids. We demonstrate, for the first time, zero-background helicase-dependent amplification (HDA), with low concentrations of both ATP and dNTPs. This strategy achieved the reliable evaluation of telomerase activity in cancer cells by eliminating Primer-Dimer artifacts, which have plagued many previous methods with reduced specificity. We found that the performance of the telomerase assay by zero-background HDA was negatively affected by highly concentrated cellular proteins. This inhibitory effect is attributed to the binding of DNA templates to proteins, thus making them unavailable for polymerases. However, gold nanoparticles were demonstrated to highly attenuate such inhibition by abundant proteins, and to enhance the assay sensitivity and reliability when the reaction was performed with concentrated cell extracts.