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Methods in Molecular Biology (Clifton,... 2023The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the...
The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.
Topics: Cloning, Molecular; DNA Ligase ATP; DNA Ligases; DNA Primers; Nucleotidyltransferases
PubMed: 36853455
DOI: 10.1007/978-1-0716-3004-4_4 -
Cold Spring Harbor Protocols Jun 2019The polymerase chain reaction (PCR) underlies almost all of modern molecular cloning. Using PCR, a defined target sequence that occurs once within a DNA of high... (Review)
Review
The polymerase chain reaction (PCR) underlies almost all of modern molecular cloning. Using PCR, a defined target sequence that occurs once within a DNA of high complexity and large size-an entire mammalian genome, for example-can be rapidly and selectively amplified in a quasi-exponential chain reaction that generates millions of copies. The reaction is simple to set up, cheap, and undemanding, the only requirement being some knowledge of the nucleotide sequences of the target. In addition to its simplicity, PCR is robust, speedy, flexible, and sensitive.
Topics: DNA Contamination; DNA Primers; Oligonucleotides; Polymerase Chain Reaction; RNA, Messenger
PubMed: 31160389
DOI: 10.1101/pdb.top095109 -
Cold Spring Harbor Protocols May 2018It is critical to determine if the ChIP actually enriched the DNA sequences that are associated with the target protein. If there are known genomic binding sites,...
It is critical to determine if the ChIP actually enriched the DNA sequences that are associated with the target protein. If there are known genomic binding sites, primers can be designed for quantitative PCR (qPCR) to determine if the known sites are specifically enriched by immunoprecipitation. If there are no known sites but candidate target genes are available, one can consider designing qPCR primers along the length of potential regulatory regions such as promoters and conserved noncoding sequences within intergenic and genic regions. If candidate target genes or potential sites are not available, ChIP-chip or ChIP-seq should be considered instead. Because real-time PCR can be performed in either a 96- or 384-well format in a minimal reaction volume and primers can be synthesized with minimal cost, ChIP-qPCR is an attractive strategy to interrogate target genes and potential regulatory regions across a large number of experimental conditions and different cell types.
Topics: Chromatin Immunoprecipitation; DNA; DNA Primers; Real-Time Polymerase Chain Reaction
PubMed: 29717044
DOI: 10.1101/pdb.prot082628 -
Cold Spring Harbor Protocols Feb 2019Nested polymerase chain reaction (PCR) is used in situations in which it is necessary to increase the sensitivity and/or specificity of PCR, for example, when amplifying...
Nested polymerase chain reaction (PCR) is used in situations in which it is necessary to increase the sensitivity and/or specificity of PCR, for example, when amplifying a particular member of a polymorphic gene family or when amplifying a cDNA copy of an mRNA present at very low abundance in a clinical specimen containing a heterogeneous population of cell types. Nested PCR usually involves two sequential amplification reactions, each of which uses a different pair of primers. The product of the first amplification reaction is used as the template for the second PCR, which is primed by oligonucleotides that are placed internal to the first primer pair. The use of two pairs of oligonucleotides allows a higher number of cycles to be performed, thereby increasing the sensitivity of the PCR. The improved specificity of the reaction derives from the binding of two separate sets of primers to the same target template. Nested PCR is an efficient method to amplify segments of long templates but requires knowledge of the sequence of the target.
Topics: DNA; DNA Primers; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 30710024
DOI: 10.1101/pdb.prot095182 -
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 -
Current Protocols in Immunology Nov 2018Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at...
Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy. Since the development of PLA in 2002, it has been increasingly used to detect the interaction between two proteins with high sensitivity and specificity. It is a simple and sensitive technique to study protein-protein interaction in cells. © 2018 by John Wiley & Sons, Inc.
Topics: Antibodies; Biological Assay; DNA Primers; Humans; Microscopy, Fluorescence; Protein Interaction Mapping
PubMed: 30238640
DOI: 10.1002/cpim.58 -
Methods in Molecular Biology (Clifton,... 2015Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and...
Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.
Topics: Algorithms; DNA Primers; Real-Time Polymerase Chain Reaction; Software
PubMed: 25697650
DOI: 10.1007/978-1-4939-2365-6_3 -
Methods in Molecular Biology (Clifton,... 2017Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed...
Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.
Topics: DNA; DNA Primers; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction
PubMed: 28540701
DOI: 10.1007/978-1-4939-7060-5_5 -
Cold Spring Harbor Protocols Jun 2019The megaprimer method is a simple and versatile approach that can be adopted to create a single mutation in a specific target region as well as to create site-specific...
The megaprimer method is a simple and versatile approach that can be adopted to create a single mutation in a specific target region as well as to create site-specific insertions, deletions, and gene fusions. This method uses three oligonucleotide primers, two rounds of polymerase chain reaction (PCR), and a DNA template containing the gene to be mutated. The first round of PCR generates a fragment with the desired mutation introduced by using one of the flanking primers and the mutant primer. This amplified fragment-the megaprimer-is used in the second PCR along with the remaining external primer to amplify a longer region of the template DNA. The final product is purified and can be cloned into an appropriate vector. By designing flanking primers with universal restriction site sequences, compatible with the vector of choice, it is possible to create different mutant clones by changing only the mutant primer. Recently, this approach has been improved by the use of forward and reverse flanking primers with significantly different melting temperatures. This allows researchers to perform both PCR steps in a single tube. This protocol has been successfully applied on templates with either low or high G + C content to amplify megaprimers 71-800 bp in length and final products ranging from 400 to 2500 bp.
Topics: DNA Primers; Gene Fusion; Mutagenesis; Mutation; Polymerase Chain Reaction
PubMed: 31160386
DOI: 10.1101/pdb.prot097824 -
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