Methylation-specific PCR: four steps in primer design

Methylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design.     

Microarray synthesis through multiple-use PCR primer design

A substantial percentage of the expense in constructing full-genome spotted microarrays comes from the cost of synthesizing the PCR primers to amplify the desired DNA. We propose a computationally-based method to substantially reduce this cost. Historically, PCR primers are designed so that each primer occurs uniquely in the genome. This condition is unnecessarily strong for selective amplification, since only the primer pair associated with each amplification need be unique. We demonstrate that careful design in a genome-level amplification project permits us to save the cost of several thousand primers over conventional approaches.     

A thermodynamic approach to PCR primer design

We developed a primer design method, Pythia, in which state of the art DNA binding affinity computations are directly integrated into the primer design process. We use chemical reaction equilibrium analysis to integrate multiple binding energy calculations into a conservative measure of polymerase chain reaction (PCR) efficiency, and a precomputed index on genomic sequences to evaluate primer specificity. We show that Pythia can design primers with success rates comparable with those of current methods, but yields much higher coverage in difficult genomic regions. For example, in RepeatMasked sequences in the human genome, Pythia achieved a median coverage of 89% as compared with a median coverage of 51% for Primer3. For parameter settings yielding sensitivities of 81%, our method has a recall of 97%, compared with the Primer3 recall of 48%. Because our primer design approach is based on the chemistry of DNA interactions, it has fewer and more physically meaningful parameters than current methods, and is therefore easier to adjust to specific experimental requirements. Our software is freely available at http://pythia.sourceforge.net.     

MPprimer: a program for reliable multiplex PCR primer design

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.     

Biologically-generated primer for PCR: PCR primer of unknown sequence.

We describe a method for producing specific PCR primers directly from PCR product, bypassing the usual need to know the primer sequence. Lack of abundance of primers derived from a PCR product is compensated for by the incorporation of an arbitrary 5'TAG sequence which acts as a surrogate template target for the bulk amplification phase. We use the technique to amplify clonospecific rearranged immunoglobulin genes, which have applications as markers of lymphoid neoplasms for tracing the success of therapy. The principle may have wider application wherever conserved and variable regions of DNA are juxtaposed.     

Homolog-specific PCR primer design for profiling splice variants

To study functional diversity of proteins encoded from a single gene, it is important to distinguish the expression levels among the alternatively spliced variants. A variant-specific primer pair is required to amplify each alternatively spliced variant individually. For this purpose, we developed a new feature, homolog-specific primer design (HSPD), in our high-throughput primer and probe design software tool, PRIMEGENS-v2. The algorithm uses a de novo approach to design primers without any prior information of splice variants or close homologs for an input query sequence. It not only designs primer pairs but also finds potential isoforms and homologs of the input sequence. Efficiency of this algorithm was tested for several gene families in soybean. A total of 187 primer pairs were tested under five different abiotic stress conditions with three replications at three time points. Results indicate a high success rate of primer design. Some primer pairs designed were able to amplify all splice variants of a gene. Furthermore, by utilizing combinations within the same multiplex pool, we were able to uniquely amplify a specific variant or duplicate gene. Our method can also be used to design PCR primers to specifically amplify homologs in the same gene family. PRIMEGENS-v2 is available at: http://primegens.org.     

Specific PCR product primer design using memetic algorithm

To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150–300 bps and 500–800 bps, and two different methods of calculating T_m for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near‐optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma‐pd/ . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Set of novel tools for PCR primer design.

We have developed a new package of computer programs and algorithms for different PCR applications, including allele-specific PCR, multiplex PCR, and long PCR. The package is included in the upcoming VectorNTI suite software and attempts to incorporate most of the current knowledge about PCR primer design. A wide range of primer characteristics is available for user manipulation to provide improved efficiency and increased flexibility of primer design. To accelerate the primer calculations, we have optimized algorithms using recent advances in computer science such as dynamic trees and lazy evaluation. Proper structural organization of input parameters provides further program acceleration. New Vector NTI primer design software allows calculations of primer pairs for long PCR amplification of 120-kb genomic DNA in 5 min under most stringent input parameters and clustering 435 primer pairs for multiplex PCR within 30 min on a standard Pentium III PC. Our program allows the user to take advantage of molecule annotation by applying different kinds of filtering features during PCR primer design.     

SSCP primer design based on single-strand DNA structure predicted by a DNA folding program.

To predict alterations in single-strand DNA mobility in non-denaturing electrophoretic gels, Zuker's RNA folding program was modified. Energy files utilized by the LRNA RNA folding algorithm were modified to emulate folding of single-strand DNA. Energy files were modified to disallow G-T base pairing. Stacking energies were corrected for DNA thermodynamics. Constraints on loop nucleotide sequences were removed. The LRNA RNA folding algorithm using the DNA fold energy files was applied to predict folding of PCR generated single-strand DNA molecules from polymorphic human ALDH2 and TPH alleles. The DNA-Fold version 1.0 program was used to design primers to create and abolish SSCP mobility shifts. Primers were made that add a 5' tag sequence or alter complementarity to an internal sequence. Differences in DNA secondary structure were assessed by SSCP analysis and compared to single-strand DNA secondary structure predictions. Results demonstrate that alterations in single-strand DNA conformation may be predicted using DNA-Fold 1.0.     

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).     

PrimerIdent: A web based tool for conserved primer design

Conserved primers across multiple species and simultaneously specific for a certain isozyme can be rare and difficult to find. PrimerIdent was developed aiming to automate this primer design and selection process in a given nucleotide sequence alignment, providing an intuitive, easy to interpret graphical result, which offers a list of all possible primers that meet the user criteria, with a colour-code identity to each sequence in the alignment. The software here presented is a simple and intuitive web based tool that is suitable for distinguishing very similar nucleotide sequences, such as isozymes-coding sequences, to enable the conserved primer design across multiple species, necessary for approaches that rely on knowing if a primer is suitable for a certain set of pre-aligned sequences, to design a specific primer to a certain sequence variation, or a combination thereof. This extremely useful software can, therefore, be used as a tool for the specific amplification of individual members of multigenic families across related species and also to evaluate the differential expression of isogenes for a given species. AVAILABILITY: http://primerident.up.pt.     

PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR

PerlPrimer is a cross-platform graphical user interface application for the design of primers for standard, bisulphite and real-time PCR, and sequencing. The program incorporates highly accurate melting-temperature and primer-dimer prediction algorithms with powerful tools such as sequence retrieval from Ensembl and the ability to BLAST search primer pairs. It aims to automate and simplify the process of primer design. AVAILABILITY: Open-source and freely available from http://perlprimer.sourceforge.net.     

Primer Prim'r: A web based server for automated primer design

The Northeast Structural Genomics Consortium (NESG) is one of nine NIH-funded pilot projects created to develop technologies needed for structural studies of proteins on a genome-wide scale. One of the most challenging aspects of this emerging field is the production of protein samples amenable to structural determination. To do this efficiently, all steps in the protein production pipeline must be automated. Here we describe the Primer program (linked from http://www-nmr.cabm.rutgers.edu/bioinformatics, www-nmr.cabm.rutgers.edu/bioinformatics, a web-based primer design program freely available to the scientific community, which was created to automate this time consuming and laborious task. This program has the ability to simultaneously calculate plasmid specific primer sets for multiple open reading frame (ORF) targets, including 96-well and greater formats. Primer includes a library of commonly used plasmid systems and possesses the ability to upload user-defined plasmid systems. In addition to calculating gene-specific annealing regions for each target, the program also adds appropriate restriction endonuclease recognition or viral recombination sites while preserving a reading frame with plasmid based fusions. Primer has several useful features such as sorting calculated primer sets by target size, facilitating interpretation of PCR amplifications by agarose gel electrophoresis, as well as supplying the molecular biologist with many important characteristics of each target such as the expected size of the PCR amplified DNA fragment and internal restriction sites. The NESG has cloned over 1500 genes using oligonucleotide primers designed by Primer.     

Novel computational methods for increasing PCR primer design effectiveness in directed sequencing

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.     

A newly designed degenerate PCR primer based on pmoA gene for detection of nitrite-dependent anaerobic methane-oxidizing bacteria from different ecological niches

A new pmoA gene-based PCR primer set was designed for detection of nitrite-dependent anaerobic oxidation of methane (n-damo) bacteria from four different ecosystems, namely rice paddy soil, freshwater reservoir, reed bed, and sludge from wastewater treatment plant. This primer set showed high specificity and efficiency in recovering n-damo bacteria from these diverse samples. The obtained sequences showed 88–94 and 90–96 % similarity to nucleotide and amino acid sequences, respectively, with the known NC10 phylum bacterium. According to the UniFrac principal coordinates analysis (PCoA), DNA sequences retrieved by the new PCR primer set in this study formed a separate group from the reported sequences, indicating higher diversity of n-damo in the environment. This newly designed PCR primer is capable of amplifying not only the currently known n-damo bacteria but also those that have not been reported, providing new information on the ecological diversity and distribution of this group of microorganisms in the ecosystem.     

Development of primer sets for PCR amplification of the PgiC gene in ferns

Polymerase chain reaction (PCR)-based nuclear DNA markers were developed for fern species. We first determined the partial nucleotide sequence of cDNA of the pgiC gene encoding cytosolic phosphoglucose isomerase from Dryopteris caudipinna, and then PCR primers for exon-primed, intron-crossing (EPIC) amplifications were designed. The EPIC primers are universally applicable to the most derived indusiate fern families such as Dryopteridaceae, Thelypteridaceae, and Woodsiaceae. The PCR products of primers 14F/16R containing two introns are moderate in size (534 bp-ca.1000 bp) and are possibly of value in phylogenetic reconstruction at specific and generic levels. Codominant nuclear DNA markers applicable to the estimation of mating systems and other population genetic studies were also developed by a combination of single-strand conformation polymorphism (SSCP) and EPIC amplification using primers 14F/15R and 15F/16R. In order to provide a case study using these markers, allelic variation of PCR products using 15F/16R was examined in populations of Arachniodes standishii (Dryopteridaceae).     

Development of primer sets for PCR amplification of the PgiC gene in ferns

Polymerase chain reaction (PCR)-based nuclear DNA markers were developed for fern species. We first determined the partial nucleotide sequence of cDNA of the pgiC gene encoding cytosolic phosphoglucose isomerase from Dryopteris caudipinna, and then PCR primers for exon-primed, intron-crossing (EPIC) amplifications were designed. The EPIC primers are universally applicable to the most derived indusiate fern families such as Dryopteridaceae, Thelypteridaceae, and Woodsiaceae. The PCR products of primers 14F/16R containing two introns are moderate in size (534 bp–ca.1000 bp) and are possibly of value in phylogenetic reconstruction at specific and generic levels. Codominant nuclear DNA markers applicable to the estimation of mating systems and other population genetic studies were also developed by a combination of single-strand conformation polymorphism (SSCP) and EPIC amplification using primers 14F/15R and 15F/16R. In order to provide a case study using these markers, allelic variation of PCR products using 15F/16R was examined in populations of Arachniodes standishii (Dryopteridaceae). Received: July 4, 2001 / Accepted: September 12, 2001