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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 -
Nature Protocols Dec 2021Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of... (Review)
Review
Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double-stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 d. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 d. The result is a genome-wide catalog of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cell Line, Tumor; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Primers; Deoxyribonucleases, Type II Site-Specific; Electroporation; Gene Editing; Genome, Human; Humans; Osteoblasts; Plasmids; Polymerase Chain Reaction; Primary Cell Culture; RNA, Guide, CRISPR-Cas Systems; T-Lymphocytes
PubMed: 34773119
DOI: 10.1038/s41596-021-00626-x -
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 -
Methods in Molecular Biology (Clifton,... 2023Amplifluor, a genotyping system used to analyze single nucleotide polymorphisms (SNPs), is supplied by Merck-Millipore. Amplifluor is based on polymerase chain reaction...
Amplifluor, a genotyping system used to analyze single nucleotide polymorphisms (SNPs), is supplied by Merck-Millipore. Amplifluor is based on polymerase chain reaction (PCR) with two competing allele-specific primers and a SNP specific common reverse primer. Sequence information flanking SNP of interest and fluorescent plate reader for end-point measurement or qPCR machine for real time measurement are required for the execution of the Amplifluor assay. In this chapter, the principle and working protocol of the Amplifluor assay based on end-point fluorescence detection of SNP allele is presented with an example.
Topics: Genotype; Polymorphism, Single Nucleotide; Polymerase Chain Reaction; DNA Primers; Alleles
PubMed: 36781643
DOI: 10.1007/978-1-0716-3024-2_13 -
Brazilian Journal of Microbiology :... Jul 2019Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be...
Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be effectively addressed, if the conventional detection methods are replaced with DNA-based molecular methods. Accordingly, it was aimed to develop a robust PCR protocol for specific detection of Salmonella spp. Out of the different primers screened, one pair of primers developed in this study targeting invA gene demonstrated 100% inclusivity for a wide range of Salmonella serotypes and 100% exclusivity for wide range of non-target species. The in silico analysis of the nucleotide sequence obtained from the PCR product suggests its potential as a hybridization probe for genus specific detection of Salmonella spp. contamination. The PCR protocol was sensitive enough to detect 15 cells per reaction using crude DNA prepared within a short time directly from artificially contaminated shrimp tissue. The study demonstrated that the result of PCR reaction can come out on the same day of sample arrival. Incorporation of this pair of primers in a multiplex PCR designed for simultaneous detection of four common seafood-borne human pathogens yielded 147 bp, 302 bp, 403 bp, and 450 bp distinct DNA bands specifically targeting E. coli, toxigenic Vibrio cholerae, Salmonella spp., and V. parahaemolyticus, respectively in a single PCR tube. The PCR methods developed in this study has the potential to be used in the seafood processing plants for effective monitoring of CCPs required for implementation of HACCP-based quality assurance system.
Topics: Animals; DNA Primers; Food Contamination; Multiplex Polymerase Chain Reaction; Palaemonidae; Salmonella; Shellfish
PubMed: 31006836
DOI: 10.1007/s42770-019-00072-8 -
Nature Communications Apr 2022One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of...
One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.
Topics: Algorithms; DNA Primers; Likelihood Functions; Multiplex Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 35410464
DOI: 10.1038/s41467-022-29500-4 -
BMC Ecology and Evolution May 2024Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for... (Comparative Study)
Comparative Study
Monitoring mollusk biodiversity is a great challenge due to their large diversity and broad distribution. Environmental DNA (eDNA) technology is increasingly applied for biodiversity monitoring, but relevant studies on marine mollusks are still limited. Although previous studies have developed several pairs of primers for mollusk eDNA analyses, most of them targeted only a small group of mollusks. In this study, seven primers were designed for the mollusk community and validated and compared with eight pairs of published primers to select the best candidates. After in silico test, MollCOI154 and MollCOI255 primers showed non-specific amplification, and same results were also obtained in published primers (COI204, Sepi, and veneroida). Moll12S100, Moll12S195 and Moll16S primers failed to amplify across all genomic DNA from selected mollusk. Except Moll16S, all developed and two published (unionoida and veneroida) primers were successfully amplified on four eDNA samples from Yangtze River estuary. After annotation of the amplified sequences, MollCOI253 showed higher annotation of the amplification results than the other primers. In conclusion, MollCOI253 had better performance in terms of amplification success and specificity, and can provide technical support for eDNA-based research, which will be beneficial for molluscan biodiversity investigation and conservation.
Topics: Mollusca; Animals; DNA Barcoding, Taxonomic; DNA, Environmental; DNA Primers; Biodiversity
PubMed: 38822255
DOI: 10.1186/s12862-024-02265-8 -
Methods in Molecular Biology (Clifton,... 2023The GlobalFiler™ PCR Amplification Kit is one of the most sensitive kits that exist today that makes the PCR amplification of human DNA possible. PCR amplification...
The GlobalFiler™ PCR Amplification Kit is one of the most sensitive kits that exist today that makes the PCR amplification of human DNA possible. PCR amplification using this specific kit makes millions of copies of 24 specific target sequences in the DNA, called markers or loci. This kit is a 6-dye, short tandem repeat (STR) multiplex assay kit that has a synthetic mix of primers and single-stranded oligonucleotides that are combined with DNA samples and then subjected to 29 or 30 cycles of denaturing, annealing, and extension, as per laboratory protocol. Methods for instrument operation will vary depending on the thermal cycler instrument model that is used. Nevertheless, the GlobalFiler™ PCR Amplification Kit has proven to be a very useful tool to DNA analysts, amplifying extremely low quantities of DNA, making it possible to detect partial, if not full, genetic profiles from a wide range of sample types. This chapter discusses the typical preparation and PCR amplification of human forensic DNA samples, using the GlobalFiler™ PCR Amplification Kit.
Topics: Humans; DNA Fingerprinting; Polymerase Chain Reaction; Microsatellite Repeats; DNA; DNA Primers
PubMed: 37439986
DOI: 10.1007/978-1-0716-3295-6_15 -
Methods in Molecular Biology (Clifton,... 2022Proximity ligation assay (PLA), also referred to as Duolink PLA technology, permits detection of protein-protein interactions in situ (<40 nm distance) at endogenous...
Proximity ligation assay (PLA), also referred to as Duolink PLA technology, permits detection of protein-protein interactions in situ (<40 nm distance) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and takes advantage of specific DNA primers covalently linked to the antibodies. A hybridization step followed by a PCR amplification with fluorescent probes then permits visualization of spots of proximity by fluorescence microscopy.
Topics: Antibodies; DNA Primers; Microscopy, Fluorescence; Protein Interaction Mapping; Proteins
PubMed: 34859407
DOI: 10.1007/978-1-0716-1948-3_13 -
PloS One 2023qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However...
AIMS
qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6.
METHODS
The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer.
RESULTS
Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition.
CONCLUSIONS
Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.
Topics: Polymerase Chain Reaction; Nucleic Acid Amplification Techniques; DNA; DNA Primers; Genomics
PubMed: 37083935
DOI: 10.1371/journal.pone.0284538