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Journal of Visualized Experiments : JoVE May 2012In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was...
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase. PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: • Set up reactions and thermal cycling conditions for a conventional PCR experiment • Understand the function of various reaction components and their overall effect on a PCR experiment • Design and optimize a PCR experiment for any DNA template • Troubleshoot failed PCR experiments.
Topics: Polymerase Chain Reaction
PubMed: 22664923
DOI: 10.3791/3998 -
The National Medical Journal of India 1992
Review
Topics: DNA Fingerprinting; Humans; Infections; Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Sensitivity and Specificity
PubMed: 1304285
DOI: No ID Found -
Journal of Clinical Laboratory Analysis 2002Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain... (Review)
Review
Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features.
Topics: Humans; Magnesium Chloride; Polymerase Chain Reaction
PubMed: 11835531
DOI: 10.1002/jcla.2058 -
The Journal of Investigative Dermatology Mar 2013
Topics: Animals; Humans; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 23399825
DOI: 10.1038/jid.2013.1 -
Trends in Biotechnology Jul 2019Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the... (Review)
Review
Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.
Topics: Costs and Cost Analysis; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Time
PubMed: 30654913
DOI: 10.1016/j.tibtech.2018.12.002 -
Journal of Applied Microbiology Nov 2012The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of... (Review)
Review
The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false-negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix-specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.
Topics: False Negative Reactions; Nucleic Acid Synthesis Inhibitors; Polymerase Chain Reaction; Quality Control; Specimen Handling
PubMed: 22747964
DOI: 10.1111/j.1365-2672.2012.05384.x -
FEMS Microbiology Ecology Jan 2009Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional... (Review)
Review
Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.
Topics: Ecology; Environmental Microbiology; Polymerase Chain Reaction; RNA, Messenger; RNA, Ribosomal, 16S; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 19120456
DOI: 10.1111/j.1574-6941.2008.00629.x -
Nucleic Acids Research Feb 2009The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction...
The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R(2) = 0.844) than with our original singleplex method (R(2) = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R(2) = 0.91).
Topics: Albumins; DNA Primers; Gene Dosage; Humans; Polymerase Chain Reaction; Reference Standards; Reproducibility of Results; Tandem Repeat Sequences; Telomere; Temperature; beta-Globins
PubMed: 19129229
DOI: 10.1093/nar/gkn1027 -
PCR Methods and Applications Dec 1993
Topics: DNA Primers; Polymerase Chain Reaction; Software
PubMed: 8118394
DOI: 10.1101/gr.3.3.s30 -
BMC Ophthalmology Oct 2016To study the value and safety of aqueous humor polymerase chain reaction (PCR) analysis for Herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr virus and...
BACKGROUND
To study the value and safety of aqueous humor polymerase chain reaction (PCR) analysis for Herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr virus and Toxoplasma gondii in patients with uveitis.
METHODS
Records of 45 consecutive patients with anterior and posterior uveitis who underwent AC paracentesis with PCR were reviewed. The main outcome measure was frequency of PCR positivity. Secondary outcomes were alteration of treatment, safety of paracentesis, and correlation of keratitic precipitates with PCR positivity, RESULTS: The overall PCR positivity was 48.9 % (22/45). Therapy was changed because of the PCR results in 14/45 patients (37.7 %). One patient experienced a paracentesis related complication (1/45, 2.2 %) without long-term sequelae.
CONCLUSION
Aqueous PCR altered the diagnosis and treatment in over a third of our patients and was relatively safe. Aqueous PCR should be considered for uveitis of atypical clinical appearance, recurrent severe uveitis of uncertain etiology, and therapy refractory cases.
Topics: Adult; Aged; Aqueous Humor; Diagnostic Techniques, Ophthalmological; Eye Infections, Viral; Female; Humans; Male; Middle Aged; Polymerase Chain Reaction; Toxoplasmosis; Uveitis; Young Adult
PubMed: 27793120
DOI: 10.1186/s12886-016-0369-z