共查询到20条相似文献,搜索用时 31 毫秒
1.
Background
Multiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step. 相似文献2.
Gang Chen Peter Kronenberger Erik Teugels Jacques De Grève 《Biological procedures online》2011,13(1):1-8
Background
Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets. 相似文献3.
Samuel Arvidsson Miroslaw Kwasniewski Diego Mauricio Riaño-Pachón Bernd Mueller-Roeber 《BMC bioinformatics》2008,9(1):465
Background
Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays. 相似文献4.
Zhiyong Shen Wubin Qu Wen Wang Yiming Lu Yonghong Wu Zhifeng Li Xingyi Hang Xiaolei Wang Dongsheng Zhao Chenggang Zhang 《BMC bioinformatics》2010,11(1):143
Background
Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. 相似文献5.
Background
Different algorithms have been proposed to solve various versions of degenerate primer design problem. For one of the most general cases, multiple degenerate primer design problem, very few algorithms exist, none of them satisfying the criterion of designing low number of primers that cover high number of sequences. Besides, the present algorithms require high computation capacity and running time. 相似文献6.
Kelvin Li Anushka Brownley Timothy B Stockwell Karen Beeson Tina C McIntosh Dana Busam Steve Ferriera Sean Murphy Samuel Levy 《BMC bioinformatics》2008,9(1):191
Background
Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment. 相似文献7.
8.
Background
Availability of genomewide information on an increasing but still limited number of plants offers the possibility of identifying orthologues, or related genes, in species with major economical impact and complex genomes. In this paper we exploit the recently described CODEHOP primer design and PCR strategy for targeted isolation of homologues in large gene families. 相似文献9.
Youfang?Cao Lianjie?Wang Kexue?Xu Chunhai?Kou Yulei?Zhang Guifang?Wei Junjian?He Yunfang?Wang Liping?Zhao
Background
A new algorithm for assessing similarity between primer and template has been developed based on the hypothesis that annealing of primer to template is an information transfer process. 相似文献10.
Background
Robust designs of PCR-based molecular diagnostic assays rely on the discrimination potential of sequence variants affecting primer-to-template annealing. However, for accurate quantitative PCR (qPCR) assessment of gene expression in populations with gene polymorphisms, the effects of sequence variants within primer binding sites must be minimized. This dichotomy in PCR applications prompted us to design experiments to specifically address the quantitative nature of PCR amplifications with oligonucleotides containing mismatches. 相似文献11.
Background
Nuclear DNA sequences provide genetic information that complements studies using mitochondrial DNA. Some 'universal' primer sets have been developed that target introns within protein-coding loci, but many simultaneously amplify introns from paralogous loci. Refining existing primer sets to target a single locus could circumvent this problem. 相似文献12.
Turnip yellow mosaic virus (TYMV) is a plant pathogenic virus transmitted mainly through its host Brassica spp. TYMV is originated from Europe. Its infection cases have been reported in Australia, Brazil, Turkey, and Japan. Symptoms similar to those of TYMV infections were also reported in Korea in 2012. In this study, we developed RT-PCR primer pairs that were highly sensitive for detecting TYMV. The developed RT-PCR primer pairs offered about 10–100 times stronger detection sensitivity compared to primer pairs previously used in Korea. As a result, a 491 bp TYMV-specific band was identified. The specific band was confirmed to be TYMV based on sequencing results and phylogenetic analysis. The RT-PCR primer pairs developed in this study can be used to rapidly and precisely diagnose TYMV in agricultural products such as Chinese cabbage and other crops infected by TYMV.
Electronic supplementary material
The online version of this article (doi:10.1007/s12088-015-0557-1) contains supplementary material, which is available to authorized users. 相似文献13.
Background
Complete mitochondrial (mt) genome sequencing is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. For long template sequencing, i.e., like the entire mtDNA, it is essential to design primers for Polymerase Chain Reaction (PCR) amplicons which are partly overlapping each other. The presented chromosome walking strategy provides the overlapping design to solve the problem for unreliable sequencing data at the 5′ end and provides the effective sequencing. However, current algorithms and tools are mostly focused on the primer design for a local region in the genomic sequence. Accordingly, it is still challenging to provide the primer sets for the entire mtDNA.Methodology/Principal Findings
The purpose of this study is to develop an integrated primer design algorithm for entire mt genome in general, and for the common primer sets for closely-related species in particular. We introduce ClustalW to generate the multiple sequence alignment needed to find the conserved sequences in closely-related species. These conserved sequences are suitable for designing the common primers for the entire mtDNA. Using a heuristic algorithm particle swarm optimization (PSO), all the designed primers were computationally validated to fit the common primer design constraints, such as the melting temperature, primer length and GC content, PCR product length, secondary structure, specificity, and terminal limitation. The overlap requirement for PCR amplicons in the entire mtDNA is satisfied by defining the overlapping region with the sliding window technology. Finally, primer sets were designed within the overlapping region. The primer sets for the entire mtDNA sequences were successfully demonstrated in the example of two closely-related fish species. The pseudo code for the primer design algorithm is provided.Conclusions/Significance
In conclusion, it can be said that our proposed sliding window-based PSO algorithm provides the necessary primer sets for the entire mt genome amplification and sequencing. 相似文献14.
Filip Pattyn Jasmien Hoebeeck Piet Robbrecht Evi Michels Anne De Paepe Guy Bottu David Coornaert Robert Herzog Frank Speleman Jo Vandesompele 《BMC bioinformatics》2006,7(1):496
Background
DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers. 相似文献15.
Background
In a SINE-based PCR assay, a primer set specific for guinea pig genome short interspersed elements DNA was used to test the utility of genomic markers for identifying the source of vertebrate blood meals of Triatoma infestans. 相似文献16.
Background
The design of oligonucleotides and PCR primers for studying large genomes is complicated by the redundancy of sequences. The eukaryotic genomes are particularly difficult to study due to abundant repeats. The speed of most existing primer evaluation programs is not sufficient for large-scale experiments.Results
In order to improve the efficiency and success rate of automatic primer/oligo design, we created a novel method which allows rapid masking of repeats in large sequence files, for example in eukaryotic genomes. It also allows the detection of all alternative binding sites of PCR primers and the prediction of PCR products. The new method was implemented in a collection of efficient programs, the GENOMEMASKER package. The performance of the programs was compared to other similar programs. We also modified the PRIMER3 program, to be able to design primers from lowercase-masked sequences.Conclusion
The GENOMEMASKER package is able to mask the entire human genome for non-unique primers within 6 hours and find locations of all binding sites for 10 000 designed primer pairs within 10 minutes. Additionally, it predicts all alternative PCR products from large genomes for given primer pairs. 相似文献17.
Background
Complex PCR applications for large genome-scale projects require fast, reliable and often highly sophisticated primer design software applications. Presently, such applications use pipelining methods to utilise many third party applications and this involves file parsing, interfacing and data conversion, which is slow and prone to error. A fully integrated suite of software tools for primer design would considerably improve the development time, the processing speed, and the reliability of bespoke primer design software applications.Results
The PD5 software library is an open-source collection of classes and utilities, providing a complete collection of software building blocks for primer design and analysis. It is written in object-oriented C++ with an emphasis on classes suitable for efficient and rapid development of bespoke primer design programs. The modular design of the software library simplifies the development of specific applications and also integration with existing third party software where necessary. We demonstrate several applications created using this software library that have already proved to be effective, but we view the project as a dynamic environment for building primer design software and it is open for future development by the bioinformatics community. Therefore, the PD5 software library is published under the terms of the GNU General Public License, which guarantee access to source-code and allow redistribution and modification.Conclusions
The PD5 software library is downloadable from Google Code and the accompanying Wiki includes instructions and examples: http://code.google.com/p/primer-design 相似文献18.
19.
Background
Enhancements in sequencing technology have recently yielded assemblies of large genomes including rat, mouse, human, fruit fly, and zebrafish. The availability of large-scale genomic and genic sequence data coupled with advances in microarray technology have made it possible to study the expression of large numbers of sequence products under several different conditions in days where traditional molecular biology techniques might have taken months, or even years. Therefore, to efficiently study a number of gene products associated with a disease, pathway, or other biological process, it is necessary to be able to design primer pairs or oligonucleotides en masse rather than using a time consuming and laborious gene-by-gene method. 相似文献20.
Akihiro Tazumi Yuki Kakinuma Naoaki Misawa John E Moore Beverley C Millar Motoo Matsuda 《BMC microbiology》2009,9(1):256