Polymerase Chain Reaction Step Research Articles

Isolation and assessment of Pseudomonas aeruginosa and Bacillus cereus as PCB-degrading bacteria in environmental bioremediation

Classical methods of treatment of polychlorinated biphenyls (PCBs) in environmental matrices such as soil encapsulation, high-temperature heating, thermal desorption, and chemical decontamination, have been costly, and they leave behind problems of environmental footprints. They must therefore give way to bioremediation techniques which are less costly and result in eco-friendly products. The aim of the study was to assess the suitability of PCB degrading bacteria (using published data) from their environmental matrix for use in environmental bioremediation. Initial isolation of the bacteria was obtained by culturing ash samples taken from the combustion chamber of an MP400 Incinerator of infectious waste at Komfo Anokye Teaching hospital (KATH) in Kumasi, Ghana, and composite soil samples taken from a transformer-servicing site at an electricity substation in Sunyani, Ghana. Test for thermotolerance was carried out by the culturing of bacteria from the soil samples and exposing them to higher temperatures (55 °C and 65 °C) to assess their resilience in an incubator for 48 h. Growing cultures were sub-cultured, after which a cocktail of bacteria isolate characterization assays was carried out on the growing isolates. These included biochemical tests on the bacterial suspensions using the analytical profile index (API 20E) for examining Enterobacteriaceae and other non-fastidious bacteria. This was followed by MALDI-ToF-MS analyses of the bacterial strains to profile and image their proteins. A molecular step was needed which was the conventional polymerase chain reaction (PCR) method, using specific primer pairs, to amplify and accentuate sections of the bacterial DNA extracts. Results of the study showed a confirmed sample site in Ghana for harvesting bacteria capable of degrading polychlorinated biphenyls (PCBs). MALDI-ToF-MS analyses revealed the possible species as Pseudomonas aeruginosa and Bacillus cereus. The conventional PCR and gel electrophoresis steps confirmed the presence of bph genes encoding the 2,3-dioxygenase enzymes in bacteria with PCB-degrading ability as documented in published data. The serial dilution of the bacterial suspensions was modified from 10−7 to 10−5 which was enough for distinct colonies to be counted, and it was a novel contribution to environmental microbiology. The confirmation of Pseudomonas and Bacillus species isolated from a transformer oil-impacted soil in this study shows promise in growing these strains in various environmental media such as in a gaseous medium to mineralize gas-phase PCBs from combustion smoke or in bottom ash residues to degrade solid-phase PCBs. It can also be applied in bioremediation of waste liquid systems containing PCBs.

Boosting D-carbamoylase activity of recombinantBacillus subtilisby adjusting gene dosage and central carbon metabolism

D-p-hydroxyphenylglycine (D-HPG) as an intermediate of semisynthetic antibiotics has an important value in the pharmaceutical industry. The high pollution and high costs of chemical synthesis make D-HPG production by biocatalysis more promising. The hydantoinase method requires D-hydantoinase and D-carbamoylase (DCase) to convert D,L-p-hydroxyphenylhydantoin (D,L-HPH) into D-HPG. The recombinantBacillus subtilisused for the whole-cell catalysis in this process needs to improve the activity and stability of DCase. The gene dosage and cell metabolism of DCase affect its activity, and this study intends to reduce the acidification effect caused by carbon catabolite repression at the genetic level. Among strains with different gene dosages, the double-copy integrated strain DN02 had the highest DCase average activity of 132 U/g dry cell weight (gDCW). When glucose was used as the carbon source, weakening glucose absorption can significantly alleviate the acidification of fermentation broth. TheglcTmutant reduced the average glucose absorption rate by about 57%, whereas the DCase activity increased to about 518 U/gDCW. In addition, modifying the CcpA-binding site incitZand the CodY-binding site incitBto increase their expression levels can also relieve the acidification of fermentation broth, which reduced the accumulation of acetate by 24% and 17%, respectively. The DCase activity of derivative strains DN16 and DN17 can reach about 615 and 641 U/gDCW. Comparing the catalytic activity of strains to dual-enzyme activities to produce D-HPG, the average whole-cell activity of strain DN17/pUBS was about fivefold higher than that of DN02/pUBS. These strategies might also be useful for other recombinant strains to express heterologous enzymes. Isolation of nucleic acids from various cells is a step of PCR. In this study, magnetic nanoparticles can be used to extract genomic DNA and total RNA due to their paramagnetism and biocompatibility. The amount and accuracy of DNA and total RNA extracted were verified by Polymerase Chain Reaction (PCR). The method has the advantages of removing dangerous reagents such as phenol and chloroform, replacing inorganic coating such as silica with organic oil, and shortening reaction time.

Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BackgroundStudies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls.ResultsThe number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups.ConclusionsDifferences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

HBN Nanoparticle-Assisted Rapid Thermal Cycling for the Detection of Acanthamoeba.

Acanthamoeba are widely distributed in the environment and are known to cause blinding keratitis and brain infections with greater than 90% mortality rate. Currently, polymerase chain reaction (PCR) is a highly sensitive and promising technique in Acanthamoeba detection. Remarkably, the rate of heating–cooling and convective heat transfer of the PCR tube is limited by low thermal conductivity of the reagents mixture. The addition of nanoparticles to the reaction has been an interesting approach that could augment the thermal conductivity of the mixture and subsequently enhance heat transfer through the PCR tube. Here, we have developed hexagonal boron nitride (hBN) nanoparticle-based PCR assay for the rapid detection of Acanthamoeba to amplify DNA from low amoeba cell density. As low as 1 × 10−4 wt % was determined as the optimum concentration of hBN nanoparticles, which increased Acanthamoeba DNA yield up to ~16%. Further, it was able to reduce PCR temperature that led to a ~2.0-fold increase in Acanthamoeba DNA yield at an improved PCR specificity at 46.2 °C low annealing temperature. hBN nanoparticles further reduced standard PCR step time by 10 min and cycles by eight; thus, enhancing Acanthamoeba detection rapidly. Enhancement of Acanthamoeba PCR DNA yield is possibly due to the high adsorption affinity of hBN nanoparticles to purine (Guanine—G) due to the higher thermal conductivity achieved in the PCR mixture due to the addition of hBN. Although further research is needed to demonstrate these findings in clinical application, we propose that the interfacial layers, Brownian motion, and percolation network contribute to the enhanced thermal conductivity effect.

Detection and Molecular Identification of Cryptosporidium Species Among Children with Malignancies

Cryptosporidiosis represents a major health problem worldwide particularly among children. Its diagnosis is still difficult and demands sensitive methods. In Egypt, there is little documentation of infection among children with malignancies. This work was designed to study the infection rate of Cryptosporidium among children with malignancies, compare the performance of modified Ziehl-Neelsen (MZN) stain with nested polymerase chain reaction (PCR) and identify the species subtypes of positive cases. The study was conducted on 100 children with malignancies (leukemia, lymphoma and solid tumors), below 10 years of age, from El-Shatby hospital, Alexandria University. After obtaining the informed consent, their stool samples were collected and examined microscopically following MZN stain for the diagnosis of Cryptosporidium spp. All samples were then subjected to nested PCR. Restriction fragment length polymorphism (RFLP) targeting the Cryptosporidium oocyst wall protein (COWP) gene was applied to positive cases, using restriction enzyme RsaI for digestion of nested PCR products. Out of the 100 examined children, MZN detected higher positive cases compared to nested PCR. Six cases (6%) were diagnosed positive by MZN stain, three of which (3%) were concordantly positive by nested PCR. All positives were among children with acute lymphoblastic leukemia (ALL). Fair agreement was found between the two tests (K = 0.36). Genotyping results revealed that positive samples were of Cryptosporidium parvum (C. parvum) type II. Low Cryptosporidium infection rate was detected among children with malignancies. MZN diagnosed more positive cases compared to nested PCR. C. parvum type II was the identified species among the examined children. Further optimization of PCR steps is needed.

Nanostructured mesoporous gold biosensor for microRNA detection at attomolar level

Advances in nanoarchitectonics enable a wide variety of nanostructured electrodes with tunable shapes and surface for constructing sensitive biosensors. Herein we demonstrate the fabrication of a mesoporous gold (Au) biosensor for the specific and sensitive detection of miRNA in a relatively simple and portable manner. The electrocatalytic activity of the mesoporous Au electrode (MPGE) towards the redox reaction of Fe(CN)6]3-/4- expansively examined. Leveraging the electrocatalytic activity and signal enhancement capacity of the MPGE, an ultrasensitive and specific electrochemical sensor was developed for the detection of microRNA (miRNA). The target miRNA from spiked samples is selectively isolated and purified using magnetic bead-capture probe followed by the direct adsorption on the MPGE through direct affinity interaction between miRNA and mesoporous Au surface. The MPGE-bound miRNA is then quantified by differential pulse voltammetry (DPV) using [Fe(CN)6]4-/3- redox system (Faradaic current decrease with reference to the bare MPGE). This method evades the cumbersome PCR (polymerase chain reaction) and enzymatic amplification steps. This is a single-step assay building which can detect a wide dynamic linear range (100 aM to 1 nM) of miRNA with an ultra-low limit detection of 100 aM and present high translational potentiality for the development of high-performance detection tools for clinics.

PCR-free paper-based nanobiosensing platform for visual detection of telomerase activity via gold enhancement

Telomerase activity has been demonstrated in a wide variety of most solid tumors and considered as a well-known cancer biomarker. The commonly utilized method for its detection is polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP). However, the TRAP technique suffers from false-negative results caused by the failure of PCR step. Moreover, it requires advanced equipment with a tedious and time-consuming procedure. Herein, we presented a portable nitrocellulose paper-based nanobiosensing platform for ultrafast and equipment-free detection of telomerase activity based on a simple colorimetric assay that enabled naked-eye visualization of the color change in response to enzyme activity. In this platform, hybridization was initially performed between telomere complementary oligonucleotide immobilized on gold nanoparticles (GNPs) and telomerase elongated biotinylated probe. Thereafter, the assembly was attached on activated paper strip via avidin-biotin interaction. The signal amplification was carried out by enlargement of the attached GNPs on the paper strip, forming tightly compact rod-shaped submicron structures of gold representing a visual color formation. Thanks to significant sensitivity enhancement, the color change was occurred for down to 6 cells, which can be easily observed by the naked eye. Due to the desired aspects of the developed assay including PCR-free, low cost, simple, and high sensitivity, it can be used for evaluation of telomerase activity in cell extracts for future clinical applications. Furthermore, this design has the ability to be easily integrated into lab-on-chip devices for point-of-care telomerase sensing.

Megaprimer Polymerase Chain Reaction (PCR)-Based Mutagenesis

The megaprimer method is a simple and versatile approach that can be adopted to create a single mutation in a specific target region as well as to create site-specific insertions, deletions, and gene fusions. This method uses three oligonucleotide primers, two rounds of polymerase chain reaction (PCR), and a DNA template containing the gene to be mutated. The first round of PCR generates a fragment with the desired mutation introduced by using one of the flanking primers and the mutant primer. This amplified fragment-the megaprimer-is used in the second PCR along with the remaining external primer to amplify a longer region of the template DNA. The final product is purified and can be cloned into an appropriate vector. By designing flanking primers with universal restriction site sequences, compatible with the vector of choice, it is possible to create different mutant clones by changing only the mutant primer. Recently, this approach has been improved by the use of forward and reverse flanking primers with significantly different melting temperatures. This allows researchers to perform both PCR steps in a single tube. This protocol has been successfully applied on templates with either low or high G + C content to amplify megaprimers 71-800 bp in length and final products ranging from 400 to 2500 bp.

Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2

BackgroundCanine parvovirus 2 (CPV-2) is one of the most common etiological agents that cause severe gastroenteritis in puppies. Early accurate diagnosis is important for infected dogs. In recent years, magnetic separation has become an efficient and useful tool for bioassays. In this study, polymerase chain reaction (PCR) combined with fluorescent lateral flow immunoassay (LFIA) based on magnetic purification assay was developed for the quantitative detection of CPV-2.ResultsThe optimum working reaction volume and reaction time for LFIA was 100 μL and 2 min, respectively. The PCR-LFIA assay only detected CPV-2, and did not show cross-detection of non-CPV strains. Experiments showed analytical sensitivity of 3 × 101 copies/μL and demonstrated the PCR-LFIA has a diagnostic agreement of 100% with conventional PCR on detection of clinical samples (22.6% positive, 14/62). Cutoff value is 146. The results were further verified by sequencing and BLAST software. The entire process from PCR step only takes ~ 80 min.ConclusionsThis approach provides an attractive platform for rapid and quantitative detection of CPV-2, indicating great promise as a convenient molecular detection tool to facilitate disease outbreak investigations and response timely.

Rapid Screening of CRISPR/Cas9-Induced Mutants Using the ACT-PCR Method.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in targeted mutagenesis. However, a significant amount of time and cost is required to screen for the CRISPR/Cas9-induced mutants from a typically large number of initial samples. Here, we describe a cost-effective and sensitive screening technique based on conventional polymerase chain reaction (PCR), termed "annealing at critical temperature PCR" (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

An Efficient Method for Purifying High-Quality RNA from the Hepatopancreas of the Pacific AbaloneHaliotis discus hannai

RNA isolation from the hepatopancreas of adult abalone is challenging, as indicated by the variable results of reverse transcription-polymerase chain reaction (RT-PCR) amplification. In this study, three RNA isolation methods were tested to evaluate their usefulness in RNA isolation from various abalone tissues. Unlike the conventional method (method I), methods II and III included additional centrifugation and lithium chloride precipitation steps, respectively. RNA quality assessment based on the RT-PCR amplification of ribosomal protein L3 (RPL3) showed little difference in the quality of RNA isolated from muscle, regardless of the isolation method used. While RNA isolated from the hepatopancreas using methods I and II resulted in lower levels of RPL3 amplification, method III was found to be a more effective method of RNA extraction from the hepatopancreas, as indicated by a 30–60-fold increase in the RPL3 level. Competitive inhibition experiments using mixtures of RNA prepared by each method were performed to investigate which RT-PCR step was inhibited by materials present in the RNA preparation. An almost complete absence of RPL3 amplification was observed in RT-PCR with a cDNA template prepared from a mixture of RNAs containing a small amount of hepatopancreatic RNA isolated using method I. A drastic decrease in the incorporation of a fluorescence-labeled nucleotide into cDNA was also evident in the reverse transcription containing hepatopancreatic RNA isolated using method I. These results indicate a distinct inhibition of cDNA synthesis by materials present in the hepatopancreatic RNA preparation isolated using method I, but not present when method III is used. A competitive inhibition experiment was performed using mixtures of cDNA prepared from RNA isolated by each method to investigate whether the materials used in the preparation inhibited the polymerase chain reaction (PCR) step. The level of RPL3 amplification was proportional to the amount of cDNA prepared from hepatopancreatic RNA isolated using method III. The comparable degree of amplification regardless of the amount of cDNA prepared from RNA isolated using method I, together with the low cDNA synthesis in the latter, indicated that it had little, if any, inhibitory effect on the PCR step. This study reports an RNA isolation method that is applicable to the hepatopancreas and other abalone tissues with similar efficacy.

An alternative DNA extraction method for detection of Blastocystis spp. in human fecal samples

Polymerase chain reaction (PCR) is an effective technique for diagnosis of Blastocystis infection. Notably, DNA isolation procedure is extremely critical for the PCR step. In the present study, a recently described extraction procedure, named as the “sand method” was modified and adapted for isolation of Blastocystis DNA. To evaluate its efficacy, the current method and QIAamp DNA Stool Mini Kit (Qiagen) were applied to fresh human stool samples. Our results indicated that, the mean DNA concentrations obtained by the sand method and the commercial kit were 48 and 55 ng/μl, respectively. Also, no DNA inhibitors were detected in two methods. The sand method was capable of detecting 16 parasites per 50 mg feces. DNA samples extracted by both methods were subjected to PCR. Blastocystis spp. were detected in 11 (31.4%) of 35 samples, and perfect agreement (κ: 1.000) was found between the PCR-sand method and PCR-commercial kit method. The samples that were detected positive by PCR-sand method were successfully sequenced, and Blastocystis subtypes (STs) were identified as ST3, ST2 and ST1. In conclusion, the present study indicates that the sand method provides a simple, rapid and inexpensive procedure for reliable extraction of Blastocystis DNA from stool samples.

Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

One of the important enzymes in cellulase complex is β-glucosidase. In this research, adding signal peptide of inulinase gene from Kluyveromyces marxianus, cloning, and expressing of bglp15.2 gene in S. cerevisiae BY4741 had been done. Gene of bglp15.2 encoding β-glucosidase has 90% identity to nucleotide sequence of Shewanella frigidimarina NCIMB 400 bacteria. Adding nucleotide sequence of signal peptide was aimed to secrete β-glucosidase and had been done with PCR (Polymerase Chain Reaction) method. The addition of nucleotide sequence of signal peptide in bglp15.2 gene had been done succesfully that indicated from nucleotide sequencing result and the increment of amplicon band size in electroferogram of the last addition PCR step. The bglp15.2 and bglp15.2INU gene (the bglp15.2 gene that has signal peptide nucleotide sequence) were cloned in Escherichia coli DH5α using pGEM-T-Easy vector and pBEVY-GL shuttle vector. The pBEVY-GL shuttle vector was used for transforming S. cerevisiae BY4741 with bglp15.2 and bglp15.2INU. The recombinant S. cerevisiae BY4741 carrying bglp15.2INU gene and growing in 48 hours had extracellularly β-glucosidase enzyme activity of 0,0178 U/ml and the intracellularly activity was 0,0181 U/ml. The β-glucosidase enzyme without signal peptide was not secreted. With K. marxianus inulinase signal peptide, about 50% Bglp15.2INU protein could be secreted. The protein molecular weight of secreted Bglp15.2INU was 44 kDa in SDS-PAGE result.

Standardization of a Two-step Real-time Polymerase Chain Reaction Based Method for Species-specific Detection of Medically Important Aspergillus Species

Purpose: Standardization of Aspergillus polymerase chain reaction (PCR) poses two technical challenges (a) standardization of DNA extraction, (b) optimization of PCR against various medically important Aspergillus species. Many cases of aspergillosis go undiagnosed because of relative insensitivity of conventional diagnostic methods such as microscopy, culture or antigen detection. The present study is an attempt to standardize real-time PCR assay for rapid sensitive and specific detection of Aspergillus DNA in EDTA whole blood. Materials and Methods: Three nucleic acid extraction protocols were compared and a two-step real-time PCR assay was developed and validated following the recommendations of the European Aspergillus PCR Initiative in our setup. In the first PCR step (pan-Aspergillus PCR), the target was 28S rDNA gene, whereas in the second step, species specific PCR the targets were beta-tubulin (for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus), gene and calmodulin gene (for Aspergillus niger). Results: Species specific identification of four medically important Aspergillus species, namely, A. fumigatus, A. flavus, A. niger and A. terreus were achieved by this PCR. Specificity of the PCR was tested against 34 different DNA source including bacteria, virus, yeast, other Aspergillus sp., other fungal species and for human DNA and had no false-positive reactions. The analytical sensitivity of the PCR was found to be 102 CFU/ml. Conclusion: The present protocol of two-step real-time PCR assays for genus- and species-specific identification for commonly isolated species in whole blood for diagnosis of invasive Aspergillus infections offers a rapid, sensitive and specific assay option and requires clinical validation at multiple centers.

A simple and efficient method for CRISPR/Cas9-induced mutant screening

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction (PCR)/restriction enzyme (RE) assay, T7 endonuclease I (T7EI) assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting (HRM) analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are time- and labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR (ACT-PCR), for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.

Analysis of Viral Epigenotypes Using Bisulfite Sequencing: A Detailed Protocol for the Crucial Bisulfite Modification and PCR Amplification Steps.

Characterization of viral DNA methylation patterns is essential to understand its function in viral pathogenesis. Bisulfite modification, followed by polymerase chain reaction (PCR) and sequencing is the most effective method for the high resolution methylation mapping of viral genomes. Since the bisulfite modification and PCR steps are the most critical ones, an optimized protocol for these two steps is presented, with special attention to potential pitfalls.

The Discovery of Rolling Circle Amplification and Rolling Circle Transcription

Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25-100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase "rolling circle" activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential ("hyperbranched") RCA amplification made possible by use of a second primer; the development of the "padlock" process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.

Micro real-time PCR device using a circulation pump

This paper reports a micro real-time polymerase chain reaction (PCR) device that is integrated with a circulation pump, heaters, and thin film thermocouple (TFTC) temperature sensors. For simple operation and easy integration, the circulation pump used to circulate a fluid in a fluid channel is made by soft-lithography. The heaters are fabricated on the bottom of a glass substrate in order to provide a uniform temperature to the reaction chambers placed in the fluid channel. The temperature in each reaction chamber is continuously monitored by TFTC temperature sensors that are integrated on the top of the glass substrate. Prior to PCR experiments, performance of the heater is extensively investigated. The spatial temperature uniformity of the three reaction chambers remains within ±1 °C of the target temperatures when voltages are applied during the PCR steps (denaturation, annealing, and extension); the temporal variation of each reaction chamber is also measured to be under ±1 °C during operation of the circulation pump. During the PCR process, the fluorescence intensity is measured in order to evaluate the efficiency of DNA amplification. The amplification efficiency of the proposed PCR device is estimated to be 85 % when compared to the fluorescence intensity obtained from a commercial PCR device.