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Cold Spring Harbor Protocols Aug 2018Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA...
Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA sequence of interest. It requires relatively little preparation compared with other mutagenesis methods and does not require the use of restriction enzymes. Because of its versatility, the method has become widely used. Unlike methods of random mutagenesis, directed mutagenesis requires that the researcher already have a specific mutation in mind to implement. Traditional overlap extension PCR mutagenesis protocols remain limited in several critical ways, especially when it comes to generating insertions and deletions. For example, traditional protocols require that all sequence alterations be embedded within the primer itself, which makes it difficult to make insertions >30 nt. This protocol describes an overlap extension PCR mutagenesis method that is more versatile than its predecessors. Using this method, one can essentially make insertions and deletions of any size at any position within a given DNA sequence. To generate an insertion mutation, first prepare an insertion fragment and two flanking fragments by PCR. In the secondary PCR, the insertion fragment is recombined with two flanking fragments derived from the original template. This method can also be used to generate deletions, which is discussed in the latter part of the protocol.
Topics: DNA Primers; Mutagenesis, Insertional; Polymerase Chain Reaction; Sequence Deletion
PubMed: 30068588
DOI: 10.1101/pdb.prot097758 -
Journal of Veterinary Diagnostic... Jul 2014The current report discusses the process in which a methods comparison study in the National Animal Health Laboratory Network is performed. Specific details are provided... (Comparative Study)
Comparative Study
The current report discusses the process in which a methods comparison study in the National Animal Health Laboratory Network is performed. Specific details are provided for designing and analyzing studies intended to evaluate analytical sensitivity, efficiency, analytical specificity, cross-contamination, repeatability, operator variability, and to compare the performance of methods using diagnostic samples. As an example, a case study is presented comparing the performance of a candidate reverse transcription polymerase chain reaction (RT-PCR) chemistry to the current RT-PCR chemistry in use when the assay was originally validated. The present study revealed that, for all of the validation factors evaluated, the candidate method performed at least as well and generally better than the current method. The candidate method was, therefore, deemed fit for the original intended purpose of the current method and rendered acceptable for use. A discussion of the case study is intended to further motivate consideration of the study designs chosen.
Topics: Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Veterinary Medicine
PubMed: 24899621
DOI: 10.1177/1040638714535402 -
Methods in Molecular Biology (Clifton,... 2023Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for...
Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for speed; sensitivity, specificity, and yield are equivalent or better than conventional PCR. What is required (and not widely available) is rapid, accurate control of reaction temperature during cycling. Specificity improves with cycling speed, and efficiency can be maintained by increasing polymerase and primer concentrations. Speed is aided by simplicity, dyes that stain double-stranded DNA are less expensive than probes, and one of the simplest polymerases, the deletion mutant KlenTaq, is used throughout. Rapid amplification can be coupled with endpoint melting analysis to verify product identity. Instead of commercial master mixes, detailed formulations for reagents and master mixes compatible with rapid cycle and extreme PCR are described.
Topics: Polymerase Chain Reaction; DNA; Temperature
PubMed: 37041449
DOI: 10.1007/978-1-0716-2950-5_14 -
Emerging Microbes & Infections Dec 2021SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens,...
SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens, including SARS-CoV-2, in nasopharyngeal specimens from individuals with symptoms of upper respiratory tract infection. We developed and validated a rapid total nucleic acid extraction method based on real-time reverse transcription-polymerase chain reaction (RT-PCR) for the reliable, high-throughput simultaneous detection of common cold viral pathogens using the BD MAX Platform. The system was evaluated using 205 nasopharyngeal swab clinical samples. For assessment of the limit of detection (LoD), we used SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) RNA standards. The BD MAX dual multiplex real-time RT-PCR panel demonstrated a sensitivity comparable to that of the World Health Organization-recommended SARS-CoV-2 assay with an LoD of 50 copies/PCR. The LoD of influenza A/B and RSV was 100-200 copies/PCR. The overall percent agreement between the BD MAX panel and laboratory-developed RT-PCR test on 55 SARS-CoV-2-positive clinical samples was 100%. Among the 55 positive cases of COVID-19 analysed, no coinfection was detected. The BD MAX rapid multiplex PCR provides a highly sensitive, robust, and accurate assay for the rapid detection of SARS-CoV-2, influenza A/B, and RSV.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; COVID-19; COVID-19 Testing; Coinfection; Female; Humans; Influenza, Human; Male; Middle Aged; Multiplex Polymerase Chain Reaction; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; Respiratory Syncytial Virus Infections; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2; Young Adult
PubMed: 33410371
DOI: 10.1080/22221751.2021.1873073 -
Human Gene Therapy Aug 2023Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of...
Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID) assay is an cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.
Topics: Humans; Dependovirus; Polymerase Chain Reaction; Genome, Viral; Real-Time Polymerase Chain Reaction
PubMed: 37276150
DOI: 10.1089/hum.2023.014 -
Analytical Biochemistry Mar 2022Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed....
Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 μl reaction volume, 2) the supply of external temperature 145°С for the denaturation zone and 0°С for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.
Topics: COVID-19; COVID-19 Nucleic Acid Testing; Convection; Humans; Polymerase Chain Reaction; RNA, Viral; SARS-CoV-2; Temperature; Time Factors
PubMed: 35074320
DOI: 10.1016/j.ab.2022.114565 -
Lab on a Chip Oct 2016The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA)... (Review)
Review
The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection. The development of isothermal nucleic acid amplification and digital PCR microfluidic devices within the last five years is also highlighted. Furthermore, we introduce various commercial microfluidic PCR devices.
Topics: Lab-On-A-Chip Devices; Polymerase Chain Reaction; Time Factors
PubMed: 27713993
DOI: 10.1039/c6lc00984k -
The Journal of Molecular Diagnostics :... Jun 2021
Topics: Biological Assay; COVID-19; Humans; Multiplex Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2
PubMed: 33866002
DOI: 10.1016/j.jmoldx.2021.04.001 -
Methods in Molecular Biology (Clifton,... 2016Real-time Apta-PCR is a methodology that can be used for a wide variety of applications ranging from food quality control to clinical diagnostics. This method takes...
Real-time Apta-PCR is a methodology that can be used for a wide variety of applications ranging from food quality control to clinical diagnostics. This method takes advantage of the combination of the sensitivity of nucleic acid amplification with the selectivity of aptamers. Ultra-low detection of target analyte can potentially be achieved, or, improved detection limits can be achieved with aptamers of low-medium affinity. Herein, we describe a generic methodology coined real-time Apta-PCR, using a model target (β-conglutin) and a competitive format, which can be adapted for the detection of any target which an aptamer has been selected for.
Topics: Aptamers, Nucleotide; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction; SELEX Aptamer Technique; Sensitivity and Specificity
PubMed: 26552825
DOI: 10.1007/978-1-4939-3197-2_14 -
Bing Du Xue Bao = Chinese Journal of... Jan 2017Digital polymerase chain reaction (dPCR) is a new method for absolute quantification of nucleic acids. The dPCR reaction solution is divided into numerous partitions... (Review)
Review
Digital polymerase chain reaction (dPCR) is a new method for absolute quantification of nucleic acids. The dPCR reaction solution is divided into numerous partitions followed by independent amplification. Target copy number is counted using statistical analyses of positive signals. in contrast to quantitative PCR, a standard curve is not necessary for dPCR. Here, we reviewed the development, principles, and applications of dPCR.
Topics: Animals; History, 20th Century; History, 21st Century; Humans; Real-Time Polymerase Chain Reaction
PubMed: 30702829
DOI: No ID Found