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Current Issues in Molecular Biology Jan 2000TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain... (Review)
Review
TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving.
Topics: Cloning, Molecular; DNA Restriction Enzymes; DNA, Recombinant; Electrophoresis, Agar Gel; Genetic Vectors; Polymerase Chain Reaction; Taq Polymerase
PubMed: 11464915
DOI: No ID Found -
Methods in Enzymology 2011Three methods for assembling multiple, overlapping DNA molecules are described. Each method shares the same basic approach: (i) an exonuclease removes nucleotides from...
Three methods for assembling multiple, overlapping DNA molecules are described. Each method shares the same basic approach: (i) an exonuclease removes nucleotides from the ends of double-stranded (ds) DNA molecules, exposing complementary single-stranded (ss) DNA overhangs that are specifically annealed; (ii) the ssDNA gaps of the joined molecules are filled in by DNA polymerase, and the nicks are covalently sealed by DNA ligase. The first method employs the 3'-exonuclease activity of T4 DNA polymerase (T4 pol), Taq DNA polymerase (Taq pol), and Taq DNA ligase (Taq lig) in a two-step thermocycled reaction. The second method uses 3'-exonuclease III (ExoIII), antibody-bound Taq pol, and Taq lig in a one-step thermocycled reaction. The third method employs 5'-T5 exonuclease, Phusion® DNA polymerase, and Taq lig in a one-step isothermal reaction and can be used to assemble both ssDNA and dsDNA. These assembly methods can be used to seamlessly construct synthetic and natural genes, genetic pathways, and entire genomes and could be very useful for molecular engineering tools.
Topics: Base Sequence; DNA; DNA Ligase ATP; DNA Ligases; Genetic Engineering; Genetic Vectors; Molecular Sequence Data; Polymerase Chain Reaction; Recombination, Genetic; Taq Polymerase
PubMed: 21601685
DOI: 10.1016/B978-0-12-385120-8.00015-2 -
BioTechniques Sep 1997By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the... (Comparative Study)
Comparative Study
By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.
Topics: Buffers; DNA Primers; Dimethyl Sulfoxide; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Glycerol; Indicators and Reagents; Magnesium Chloride; Nucleotides; Polymerase Chain Reaction; Serum Albumin, Bovine; Solutions; Taq Polymerase; Temperature; Templates, Genetic; Time Factors
PubMed: 9298224
DOI: 10.2144/97233rr01 -
Biochemistry Dec 2020Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR....
Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.
Topics: Animals; COVID-19; Humans; Mice; Moloney murine leukemia virus; RNA-Directed DNA Polymerase; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2; Taq Polymerase
PubMed: 33275410
DOI: 10.1021/acs.biochem.0c00778 -
Proceedings of the National Academy of... Mar 2022Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current...
Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.
Topics: Chromosome Deletion; Chromosomes, Human, Y; DNA Primers; Escherichia coli; Flap Endonucleases; Fluorescent Dyes; Humans; Limit of Detection; Multiplex Polymerase Chain Reaction; Taq Polymerase
PubMed: 35197282
DOI: 10.1073/pnas.2110672119 -
Science Advances Mar 2022Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here,...
Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq's fingers domain testing complementarity and orientation. On average, one transient closure was observed for every nucleotide binding event; even complementary substrate pairs averaged five transient closures between each catalytic incorporation at 72°C. The rate and duration of the transient closures and the catalytic events had almost no temperature dependence, leaving all of Taq's temperature sensitivity to its rate-determining open state.
Topics: Catalysis; DNA Replication; Kinetics; Nucleotides; Taq Polymerase
PubMed: 35275726
DOI: 10.1126/sciadv.abl3522 -
Nucleic Acids Research 2004Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. Here we...
Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. Here we report a protein engineering-based approach to significantly improve the processivity of DNA polymerases by covalently linking the polymerase domain to a sequence non-specific dsDNA binding protein. Using Sso7d from Sulfolobus solfataricus as the DNA binding protein, we demonstrate that the processivity of both family A and family B polymerases can be significantly enhanced. By introducing point mutations in Sso7d, we show that the dsDNA binding property of Sso7d is essential for the enhancement. We present evidence supporting two novel conclusions. First, the fusion of a heterologous dsDNA binding protein to a polymerase can increase processivity without compromising catalytic activity and enzyme stability. Second, polymerase processivity is limiting for the efficiency of PCR, such that the fusion enzymes exhibit profound advantages over unmodified enzymes in PCR applications. This technology has the potential to broadly improve the performance of nucleic acid modifying enzymes.
Topics: Amino Acid Sequence; Archaeal Proteins; Buffers; Catalysis; DNA; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Enzyme Stability; Molecular Sequence Data; Point Mutation; Polymerase Chain Reaction; Potassium Chloride; Protein Engineering; Recombinant Fusion Proteins; Sequence Deletion; Taq Polymerase
PubMed: 14973201
DOI: 10.1093/nar/gkh271 -
Microbiology (Reading, England) Jan 2002The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by...
The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey's Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.
Topics: Bacteria, Aerobic; Bacteria, Anaerobic; Bacterial Infections; Colony Count, Microbial; Culture Media; DNA Probes; DNA, Bacterial; DNA, Ribosomal; Dental Caries; Dentin; Gene Dosage; Humans; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Taq Polymerase
PubMed: 11782518
DOI: 10.1099/00221287-148-1-257 -
BioTechniques Jul 2000
Topics: Aging; Animals; Buffers; DNA; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Hot Temperature; Hydrogen-Ion Concentration; Indicators and Reagents; Magnesium Chloride; Mice; Polymerase Chain Reaction; Sodium Hydroxide; Solubility; Taq Polymerase; Thymine Nucleotides
PubMed: 10907076
DOI: 10.2144/00291bm09 -
The Journal of Molecular Diagnostics :... Nov 2022Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the...
Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.
Topics: Humans; Taq Polymerase; Alleles; DNA; DNA Primers; Polymerase Chain Reaction
PubMed: 36058471
DOI: 10.1016/j.jmoldx.2022.08.002