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BioMed Research International 2021This study presents a discussion on the fundamentals of polymerase chain reaction (PCR) and its use as a diagnostic tool in periodontology. (Review)
Review
OBJECTIVES
This study presents a discussion on the fundamentals of polymerase chain reaction (PCR) and its use as a diagnostic tool in periodontology.
MATERIALS AND METHODS
A computer-aided as well as hand-made search in PubMed and Scopus indexed journals (relevant to the topic) was done by keywords of molecular technique in periodontology, PCR, applications of PCR, and PCR in periodontics. Only the papers in the English language and outlining PCR and its association with periodontology were collected and utilized to provide a succinct review. There was no limitation for publication time.
RESULTS
The results of our search showed that PCR has turned into a standard in diagnosis in the field of periodontology. A variety of researches has demonstrated that its sensitive, and specific characteristics make it a quick and effective technique of recognition, identification, and quantification of microorganisms. Identification of various immunoinflammatory markers at the mRNA expression level as well as ascertaining gene-related polymorphisms can also be performed.
CONCLUSIONS
The mechanisms of periodontal disease can further become clarified using PCR. . PCR as a diagnostic method can play a main part in the validation of the clinical diagnosis of periodontal disease indicating the reason, pathogenesis, clinical steps, progress, and prognosis of the disease.
Topics: Biomarkers; Humans; Molecular Diagnostic Techniques; Periodontal Diseases; Periodontics; Polymerase Chain Reaction; Polymorphism, Genetic
PubMed: 34337068
DOI: 10.1155/2021/9979948 -
Cytotherapy Jan 2023Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be...
BACKGROUND AIMS
Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct.
METHODS
The authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer-probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct.
RESULTS
ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors' assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities.
CONCLUSIONS
The authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test-retest reliability, and that specific assays can be developed for detection of unique constructs.
Topics: Humans; Reproducibility of Results; Receptors, Chimeric Antigen; DNA Copy Number Variations; T-Lymphocytes; Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction
PubMed: 36253252
DOI: 10.1016/j.jcyt.2022.09.004 -
Molecular Oncology May 2023Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many...
Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.
Topics: Humans; Reverse Transcriptase Polymerase Chain Reaction; Reproducibility of Results; RNA; Real-Time Polymerase Chain Reaction
PubMed: 36916500
DOI: 10.1002/1878-0261.13418 -
BioTechniques Jul 2019
Topics: Animals; Equipment Design; Humans; Miniaturization; Polymerase Chain Reaction; Time Factors
PubMed: 31238703
DOI: 10.2144/btn-2019-0076 -
Molecular Biotechnology Apr 2022The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent... (Review)
Review
The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.
Topics: Biosensing Techniques; COVID-19 Testing; CRISPR-Cas Systems; Electrochemical Techniques; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Molecular Diagnostic Techniques; Neutralization Tests; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; SARS-CoV-2; Surface Plasmon Resonance
PubMed: 34655396
DOI: 10.1007/s12033-021-00417-5 -
Biophysical Journal Apr 2023The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent...
The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent applications, including virus tests, DNA sequencing, genotyping, and genome cloning. These applications rely on the specificity of the primer hybridization and therefore require effective suppression of hybridization errors. A simple and effective method to achieve that is to add blocker strands, also called clamps, to the PCR mixture. These strands bind to the unwanted target sequence, thereby blocking the primer mishybridization. Because of its simplicity, this method is applicable to a broad nucleic-acid-based biotechnology. However, the precise mechanism by which blocker strands suppress PCR errors remains to be understood, limiting the applicability of this technique. Here, we combine experiments and theoretical modeling to reveal this mechanism. We find that the blocker strands both energetically destabilize the mishybridized complex and sculpt a kinetic barrier to suppress mishybridization. This combination of energetic and kinetic biasing extends the viable range of annealing temperatures, which reduces design constraint of the primer sequence and extends the applicability of PCR.
Topics: DNA Primers; Polymerase Chain Reaction; Nucleic Acid Hybridization; Nucleic Acids; Temperature
PubMed: 36823986
DOI: 10.1016/j.bpj.2023.02.028 -
Parasites & Vectors Nov 2022Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world's poorest regions. Transmitted by the bite... (Review)
Review
Leishmania infections span a range of clinical syndromes and impact humans from many geographic foci, but primarily the world's poorest regions. Transmitted by the bite of a female sand fly, Leishmania infections are increasing with human movement (due to international travel and war) as well as with shifts in vector habitat (due to climate change). Accurate diagnosis of the 20 or so species of Leishmania that infect humans can lead to the successful treatment of infections and, importantly, their prevention through modelling and intervention programs. A multitude of laboratory techniques for the detection of Leishmania have been developed over the past few decades, and although many have drawbacks, several of them show promise, particularly molecular methods like polymerase chain reaction. This review provides an overview of the methods available to diagnostic laboratories, from traditional techniques to the now-preferred molecular techniques, with an emphasis on polymerase chain reaction-based detection and typing methods.
Topics: Animals; Humans; Female; Leishmaniasis; Psychodidae; Leishmania; Polymerase Chain Reaction; Phlebotomus
PubMed: 36335408
DOI: 10.1186/s13071-022-05524-z -
Scientific Reports Jul 2023The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination...
The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA's integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.
Topics: DNA, Complementary; Reverse Transcription; Polymerase Chain Reaction; DNA; RNA; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 37454173
DOI: 10.1038/s41598-023-38383-4 -
Biosensors May 2022Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30−40 min, and this time is...
Polymerase chain reaction (PCR) is limited by the long reaction time for point-of-care. Currently, commercial benchtop rapid PCR requires 30−40 min, and this time is limited by the absence of rapid and stable heating and cooling platforms rather than the biochemical reaction kinetics. This study develops an ultrafast PCR (<3 min) platform using flow-through microchannel chips. An actin gene amplicon with a length of 151 base-pairs in the whole genome was used to verify the ultrafast PCR microfluidic chip. The results demonstrated that the channel of 56 μm height can provide fast heat conduction and the channel length should not be short. Under certain denaturation and annealing/extension times, a short channel design will cause the sample to drive slowly in the microchannel with insufficient pressure in the channel, causing the fluid to generate bubbles in the high-temperature zone and subsequently destabilizing the flow. The chips used in the experiment can complete 40 thermal cycles within 160 s through a design with the 56 µm channel height and with each thermal circle measuring 4 cm long. The calculation shows that the DNA extension speed is ~60 base-pairs/s, which is consistent with the theoretical speed of the Klen Taq extension used, and the detection limit can reach 67 copies. The heat transfer time of the reagent on this platform is very short. The simple chip design and fabrication are suitable for the development of commercial ultrafast PCR chips.
Topics: DNA; Microfluidics; Polymerase Chain Reaction
PubMed: 35624604
DOI: 10.3390/bios12050303 -
International Journal of Molecular... Jul 2023DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an...
DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing DNA polymerase for routine use in laboratories. We developed a method using () that expresses thermostable DNA polymerase directly in the PCR without purification. The gene was transformed into and expressed. After overnight incubation and washing, -expressing DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.
Topics: Taq Polymerase; Escherichia coli; Polymerase Chain Reaction; DNA; DNA Replication
PubMed: 37511160
DOI: 10.3390/ijms241411405