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Bioinformatics (Oxford, England) Nov 2002DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific...
MOTIVATION
DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications.
RESULTS
MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.
Topics: Base Sequence; Computer-Aided Design; CpG Islands; DNA Methylation; DNA Primers; Equipment Design; Estrogen Receptor beta; Humans; Internet; Molecular Sequence Data; Multimedia; Pilot Projects; Polymerase Chain Reaction; Quality Control; Receptors, Estrogen; Sensitivity and Specificity; Sequence Alignment; Sequence Analysis, DNA; Software; User-Computer Interface
PubMed: 12424112
DOI: 10.1093/bioinformatics/18.11.1427 -
Cold Spring Harbor Protocols May 2018"Touchdown polymerase chain reaction (PCR)" is a method to decrease off-target priming and hence to increase the specificity of PCRs. In touchdown PCR the temperature...
"Touchdown polymerase chain reaction (PCR)" is a method to decrease off-target priming and hence to increase the specificity of PCRs. In touchdown PCR the temperature selected for the annealing step is initially set 5°C-10°C higher than the calculated of the primers. Annealing under conditions of high stringency favors the formation of perfect primer-template hybrids. In subsequent cycles, the annealing temperature is gradually decreased by a small amount so that by the end of the PCR, the annealing temperature is 2°C-5°C below the calculated of the primers. By then, the target sequence will have undergone several cycles of geometric amplification and therefore becomes the dominant product of the PCR. To minimize mispriming during the early stages of the PCR, touchdown PCR should always be performed in conjunction with a hot start protocol. The use of touchdown PCR is essential when the sequence of the primer might not match that of the target-for example, if the sequence of the primer has been deduced from amino acid sequences, when the template DNA may contain several closely related targets, or when the target DNA is of a different species from that used to design the primers.
Topics: DNA; DNA Primers; Indicators and Reagents; Polymerase Chain Reaction
PubMed: 29717053
DOI: 10.1101/pdb.prot095133 -
Methods in Enzymology 2013Site-directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study the relative...
Site-directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study the relative importance of a particular amino acid for protein structure and function. Typical mutations are designed to disrupt or map protein-protein interactions, mimic or block posttranslational modifications, or to silence enzymatic activity. Alternatively, noncoding changes are often used to generate rescue constructs that are resistant to knockdown via RNAi.
Topics: DNA Primers; Escherichia coli; Genetic Vectors; Mutagenesis, Site-Directed; Mutation; Polymerase Chain Reaction
PubMed: 24011050
DOI: 10.1016/B978-0-12-418687-3.00019-7 -
Methods in Molecular Biology (Clifton,... 2010Primer design is a crucial initial step in any experiment utilizing PCR to target and amplify a known nucleotide sequence of interest. Properly designed primers will...
Primer design is a crucial initial step in any experiment utilizing PCR to target and amplify a known nucleotide sequence of interest. Properly designed primers will increase PCR amplification efficiency as well as isolate the targeted sequence of interest with higher specificity. Many factors that may limit the success of a primer pair can be detected a priori with computational methods. For example, primer dimer detection, amplification of alternative products, stem loop interference, extreme melting temperatures, and genotype-specific variations in the target sequence can all be considered computationally to minimize subsequent PCR failures. The use of computational sequence analysis tools to select the best primer pair from the available candidates will not only reduce experimental rates of failure but also avoid the generation of misleading results arising from the amplification of alternative products.
Topics: Amino Acid Sequence; Base Sequence; Computational Biology; DNA Primers; Databases, Genetic; Molecular Sequence Data; Nucleic Acid Conformation; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; Sequence Analysis, DNA
PubMed: 20301004
DOI: 10.1007/978-1-60761-629-0_18 -
Methods in Molecular Biology (Clifton,... 2020Primers are critical components of any PCR assay, as they are the main determinants of its specificity, sensitivity, and robustness. Despite the publication of numerous...
Primers are critical components of any PCR assay, as they are the main determinants of its specificity, sensitivity, and robustness. Despite the publication of numerous guidelines, the actual design of many published assays is often unsound: primers lack the claimed specificity, they may have to compete with secondary structures at their binding sites, primer dimer formation may affect the assay's sensitivity or they may bind only within a narrow temperature range. This chapter provides simple guidance to avoid these most common issues.
Topics: Binding Sites; DNA Primers; Limit of Detection; Nucleic Acid Conformation; Polymerase Chain Reaction; Reproducibility of Results; Temperature
PubMed: 31578684
DOI: 10.1007/978-1-4939-9833-3_2 -
Annual Review of Biochemistry 2001DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA... (Review)
Review
DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases. Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. Most DNA primases can be divided into two classes. The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases. These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase. The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit. The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle.
Topics: DNA Primers; Templates, Genetic
PubMed: 11395402
DOI: 10.1146/annurev.biochem.70.1.39 -
Methods in Molecular Biology (Clifton,... 2015Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the...
Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like "Primer3" and "Web Primer". For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment.
Topics: DNA Primers; Polymerase Chain Reaction
PubMed: 25697657
DOI: 10.1007/978-1-4939-2365-6_10 -
Cold Spring Harbor Protocols Feb 2019The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates...
The efficiency of polymerase chain reaction (PCR) amplification is influenced by the nucleotide composition and sequence of the template DNA. Problematic templates include those with long homopolymeric runs, inverted repeats, or GC-rich tracts-such as those containing >60% G + C residues-that are found in the regulatory regions of many mammalian genes. Localized regions of templates rich in GC residues tend to fold into complex secondary structures that might not melt during the annealing phase of the PCR cycle. Also, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Predictably, amplification of full-length template DNA is inefficient, and the products of the reaction contain a high proportion of shorter molecules that result from blockage of the DNA polymerase. Altering the design of the primers and using a combination of hot start and touchdown PCR can sometimes improve the efficiency of amplification. More often, a multipronged approach is required, such as the use of enhancers in the amplification reaction, adjustment of the cycling protocol, and, if necessary, designing new sets of primers. This protocol uses a mixture of four additives-betaine, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), and bovine serum albumin (BSA)-for use with DNA polymerase.
Topics: Base Composition; Buffers; DNA; DNA Primers; Nucleic Acid Conformation; Polymerase Chain Reaction; Taq Polymerase
PubMed: 30710022
DOI: 10.1101/pdb.prot095141 -
Methods in Molecular Biology (Clifton,... 2015Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional...
Quantitative real-time polymerase chain reaction (qPCR) is a powerful tool for analysis and quantification of gene expression. It is advantageous compared to traditional gel-based method of PCR, as gene expression can be visualized "real-time" using a computer. In qPCR, a reporter dye system is used which intercalates with DNA's region of interest and detects DNA amplification. Some of the popular reporter systems used in qPCR are the following: Molecular Beacon(®), SYBR Green(®), and Taqman(®). However, success of qPCR depends on the optimal primers used. Some of the considerations for primer design are the following: GC content, primer self-dimer, or secondary structure formation. Freely available software could be used for ideal qPCR primer design. Here we have shown how to use some freely available web-based software programs (such as Primerquest(®), Unafold(®), and Beacon designer(®)) to design qPCR primers.
Topics: DNA Primers; Real-Time Polymerase Chain Reaction; Software
PubMed: 25697660
DOI: 10.1007/978-1-4939-2365-6_13 -
PCR Methods and Applications Dec 1993
Topics: DNA Primers; Polymerase Chain Reaction; Software
PubMed: 8118394
DOI: 10.1101/gr.3.3.s30