Polymerase Chain Reaction Tube Research Articles

A microfluidic-based quantitative analysis system for the multiplexed genetic diagnosis of human viral infections using colorimetric loop-mediated isothermal amplification.

In this study, a microfluidic-based system utilizing colorimetric loop-mediated isothermal amplification (LAMP) is introduced for the quantitative analysis of nucleic acid targets. This system offers a user-friendly and cost-effective platform for the multiplexed genetic diagnosis of various infectious diseases across multiple samples. It includes time-lapse imaging equipment for capturing images of the microfluidic device during the LAMP assay and a hue-based quantitative analysis software to analyze the LAMP reaction, streamlining diagnostic procedures. An electric pipette was used to simplify the loading of samples and LAMP reagents into the device, allowing easy operation even by untrained individuals. The hue-based analysis software employs efficient image processing and post-processing techniques to calculate DNA amplification curves based on color changes in multiple reaction chambers. This software automates several tasks, such as identifying reaction chamber areas from time-lapse images, quantifying color information within each chamber, correcting baselines of DNA amplification curves, fitting experimental data to theoretical curves, and determining the threshold time for each curve. To validate the developed system, conventional off-chip LAMP assays were conducted with a 25 μL reaction mixture in 0.2 mL polymerase chain reaction (PCR) tubes using a real-time turbidimeter. The results indicated that the threshold time obtained using the colorimetric LAMP assay in the developed system is comparable to that obtained with real-time turbidity measurements in PCR tubes, demonstrating the system's capability for quantitative analysis of target nucleic acids, including those from human herpesviruses.

Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

An idea to explore: Determination of single nucleotide polymorphisms in alcohol metabolism-related genes using PCR-based assays to understand the link between an individual's genotype and phenotype.

Here, we propose a laboratory exercise to quickly determine single nucleotide polymorphisms (SNPs) in human alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes involved in alcohol metabolism. In this exercise, two different genotyping methods based on polymerase chain reaction (PCR), namely allele-specific (AS) PCR and a PCR-restriction fragment polymorphism (RFLP) analysis, can be performed under the same PCR program (2-step × 35 cycles, 35 min total) in parallel using a hair root lysate as a template. In AS-PCR, the target regions of the G- or A-alleles of both genes are allele-specifically amplified in a single PCR tube. In the PCR-RFLP analysis, the two genes are amplified simultaneously in a single tube, and then a portion of the PCR product is double-digested with restriction enzymes MslI and Eam1104I for 5 min. The resulting reaction products of each method are electrophoresed side by side, and the genotypes are determined from the DNA band patterns. With the optimized protocol, the whole process from template preparation to genotyping can be completed in about 75 min. During PCR, students also perform an ethanol patch test to estimate their ability to metabolize alcohol. This series of experiments can help students learn the principles and applications of PCR/SNP analyses. By comparing the genotypes revealed by PCR and the phenotypes revealed by the patch tests, students can gain a better understanding of the clinical value of genetic testing.

Portable Heating System Based on a Liquid Metal Bath for Rapid PCR

With the outbreak of COVID-19 around the world, rapid and accurate detection of new coronaviruses is the key to stop the transmission of the disease and prevent and control the novel coronavirus, among which polymerase chain reaction (PCR) is the mainstream nucleic acid detection method. A temperature cycling device is the core of the PCR amplification micro-device. The precision of the temperature control and temperature change rate directly affect the efficiency of PCR amplification. This study proposes a new PCR method based on rapid PCR chip optimization of a liquid metal bath, which realizes precise and rapid temperature rise and fall control. We systematically explored the feasibility of using liquid metals with different melting points in the system and proposed a 47 °C bismuth-based liquid metal bath as the heat conduction medium of the system to optimize the system. The heat conduction properties of the thermally conductive silicone oil bath were compared. Compared with the thermally conductive silicone oil bath, thermal cycle efficiency is improved nearly 3 times. The average heating rate of the liquid metal bath is fast, and the temperature control stability is good, which can significantly reduce the hysteresis, and the temperature change curve is more gentle, which can greatly improve the efficiency of PCR amplification. The results of gene amplification using rat DNA as the template and SEC61A as the target also indicate that the system can be successfully used in PCR devices, and the types of PCR containers can be not limited to PCR tubes. Based on the experiment, we proved that the PCR method optimized by the liquid metal bath multi-gene rapid PCR chip can further improve the temperature response speed. It has the advantages of accurate data, fast response speed, low price, safety, and environmental protection and can effectively reduce the time of PCR and improve the application efficiency. As far as we know, this is the first international report on using a liquid metal bath to do rapid-cooling PCR.

Understanding disposable plastics effects generated from the PCR testing labs during the COVID-19 pandemic

In medical labs, especially in polymerase chain reaction (PCR) testing labs, plastic residues (PCR tubes, pipet tips, falcon tubes, buffer bottles, medical globes, and others) wastes are potential sources of plastic waste. Evidence showed that a single PCR test for COVID-19 diagnosis used 37 g of disposable plastic per sample. Globally, an estimated amount of above 15,000 tons of plastic residue have been generated from the PCRs tests during the COVID-19 pandemic. These plastic residues are mismanaged and dumped with other solid wastes, especially in molecular testing labs (MTLs) from academic institutes such as universities thereby polluting the ecosystem. Plastic wastes from PCR testing labs also contain hazardous chemicals and pathogenic microorganisms. Thus, plastic residues in PCR testing labs are an important add-on source to conventional plastic wastes. In this perspective, research questions on (1) type and characteristics of plastic, (2) quantity of plastic residues as an add-on source to the conventional plastic wastes, (3) prevalence of microplastics generated from PCR testing labs of plastic wastes, (4) handling, disinfection techniques, and management strategies of these plastic residues, (5) PCR test materials as a source of hazardous chemical pollutants, and (6) future environmental pollution threats imposed by genetic material determination were raised. It is suggested that this work will be used as the baseline information in addressing the knowledge gap for improving PCR testing labs plastic waste management, and regulation to control environmental pollution. Understanding these plastics' impacts and risks is crucial for driving predictions and innovative technology processes towards sustainability.

Cryopreservation of vegetative cells and zygotes of the multicellular volvocine green alga Gonium pectorale

BackgroundColonial and multicellular volvocine green algae have been extensively studied recently in various fields of the biological sciences. However, only one species (Pandorina morum) has been cryopreserved in public culture collections.ResultsHere, we investigated conditions for cryopreservation of the multicellular volvocine alga Gonium pectorale using vegetative colonies or cells and zygotes. Rates of vegetative cell survival in a G. pectorale strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3% and 6%) of the cryoprotectant N,N-dimethylformamide (DMF) and two types of tubes (0.2-mL polymerase chain reaction tubes and 2-mL cryotubes) used for cryopreservation. Among the four conditions investigated, the highest rate of survival [2.7 ± 3.6% (0.54–10%) by the most probable number (MPN) method] was obtained when 2.0-mL cryotubes containing 1.0 mL of culture samples with 6% DMF were subjected to cryogenic treatment. Using these optimized cryopreservation conditions, survival rates after freezing in liquid nitrogen were examined for twelve other strains of G. pectorale and twelve strains of five other Gonium species. We obtained ≥ 0.1% MPN survival in nine of the twelve G. pectorale strains tested. However, < 0.1% MPN survival was detected in eleven of twelve strains of five other Gonium species. In total, ten cryopreserved strains of G. pectorale were newly established in the Microbial Culture Collection at the National Institute for Environmental Studies. Although the cryopreservation of zygotes of volvocine algae has not been previously reported, high rates (approximately 60%) of G. pectorale zygote germination were observed after thawing zygotes that had been cryopreserved with 5% or 10% methanol as the cryoprotectant during two-step cooling and freezing in liquid nitrogen.ConclusionsThe present study demonstrated that cryopreservation of G. pectorale is possible with 6% DMF as a cryoprotectant and 1.0-mL culture samples in 2.0-mL cryotubes subjected to two-step cooling in a programmable freezer.

A versatile assay platform for enzymatic poly(ethylene-terephthalate) degradation.

Accumulation of plastic and subsequent microplastic is a major environmental challenge. With the discovery of potent polyethylene terephthalate (PET)-degrading enzymes, a new perspective arose for environmental decomposition as well as technical recycling. To explore the enormous diversity of potential PET-degrading enzymes in nature and also to conveniently employ techniques like protein engineering and directed evolution, a fast and reliable assay platform is needed. In this study we present our versatile solution applying a PET coating on standard lab consumables such as polymerase chain reaction tubes, 96- and 384-well microtiter plates, yielding an adjustable crystallinity of the PET. Combining the reaction vessels with either ultra-high performance liquid chromatography (UHPLC) or fluorometric readout and additional enzyme quantification offers a range of advantages. Thereby, the platform can easily be adapted to diverse needs from detailed analysis with high precision to high-throughput (HT) applications including crude lysate analysis.

All‐in‐One DNA Extraction Tube for Facilitated Real‐Time Detection of Infectious Pathogens

An "all-in-one tube" platform is developed, where the genetic analysis involving DNA extraction, amplification, and detection can be performed in a single tube. The all-in-one tube consists of a polymerase chain reaction (PCR) tube in which the inner surface is conformally modified with a tertiary-amine-containing polymer to generate a strong electrostatic interaction with DNA. The all-in-one tube provides high DNA capture efficiency exceeding 80% from Escherichia coli O157: H7 pathogen at a wide range of DNA amount from 0.003 to 3ng. Indeed, the use of the surface-functionalized PCR tube enables direct amplification and detection of the surface-captured DNA without the modification of standard real-time PCR instrument. Besides, this platform has sensitivity, selectivity, and reliability enough for accurate detection at the minimal infective dose of both gram-positive and negative pathogens. The all-in-one tube enables the direct molecular diagnosis, substantially reducing the labor-intensive pathogen detection steps while providing high compatibility with the currently established real-time PCR instruments, and illustrates its on-site applicability with convenience expandable to various genetic analyses including food safety testing, forensic analysis, and clinical diagnosis.

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings.

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Rapid visual detection of FecB gene expression in sheep.

Sheep play an important role in agricultural production and people’s lives, and fecundity is one of the most important economic traits of sheep for sheep breeders. The Booroola fecundity (FecB) gene has a certain correlation with litter size in sheep. Therefore, this study aims to detect FecB expression quickly, accurately and visually. Here, we used the nucleic acid dye SYBR Green I to detect FecB with the amplification refractory mutation system (ARMS), which can efficiently, rapidly, economically and visually detect FecB expression in sheep. After ARMS polymerase chain reaction (PCR), SYBR Green I was directly added to the ARMS products, and whether the sheep carried FecB was judged by directly observing the color change in the PCR tube. Homozygous (BB) or heterozygous (B+) samples with FecB mutation were bright green, while wild type (++) samples without FecB were orange yellow. This study suggested that this method has 100% accuracy and 0.5 ng/µL sensitivity. To our knowledge, this is the first report that shows the integration of the ARMS with SYBR Green I to detect FecB expression in sheep visually.

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.

Sealed culture system for supporting mouse preimplantation embryo development in vitro.

This study investigated the possibility of a sealed culture system in polymerase chain reaction (PCR) tubes to maintain embryo development. The embryo density that could support the development of 2-cell stage mouse embryos to the hatching stage was determined. At an embryo density of 1:2 (100 embryos cultured in 200μL CZB medium that had been pretreated with a reference gas containing 5% O2), the developmental rate was higher and fewer embryos exhibited reactive oxygen species- or hypoxia-induced injury compared with other sealed culture groups. Expression of growth factors (insulin-like growth factor (IGF) 1, IGF2, epidermal growth factor and transforming growth factor-α) and their receptors was evaluated, with similar expression patterns seen for embryos in sealed culture (5% O2, embryo density of 1:2) compared with the control group (embryos cultured in microdrops and placed in a 37°C, 5% CO2 water-jacketed incubator; P>0.05). After transfer of blastocysts generated by the sealed culture into recipients, there were no obvious differences in the rate of normal live pups births between the sealed culture and control groups (P>0.05). Thus, the sealed embryo culture system in PCR tubes is feasible for use in situations which cannot use a traditional incubator, such as in space and during the transport of embryos.

A comparison in precision and accuracy of the conventional syringe to the Suh precision syringe.

PurposeTo compare the accuracy and precision of a conventional intraocular syringe to the Suh precision syringe (SPS), a new injection device designed to decrease operator error and to reduce adverse outcomes by providing a more ergonomic technique for periocular and intraocular injections.MethodsParticipants were instructed to inject a specified quantity of water into a 0.2 mL polymerase chain reaction (PCR) tube and a sheep eye, first with the conventional syringe, and then with the SPS. The degree of needle movement during injection was video recorded and analyzed for forward-retraction movement and variability in movement. The volume of water injected into the PCR tube was also documented. Data was analyzed using a Wilcoxon signed-rank test.ResultsAs compared to the conventional syringe, the SPS had significantly less forward-retraction movement when injecting into the sheep eye (P=0.04). Similarly, the forward-retraction movement of the needle when injecting into the PCR tube was less while using the SPS compared with the conventional syringe; however, this was not statistically significant (P=0.09). There was no significant difference in the volume of water expelled from both syringes (P=0.28).ConclusionThe SPS demonstrates significant potential to decrease the overall risk involved with injections through reduction of forward-retraction movement during device operation. This new syringe design also provides better control with injection depth and drug injection volume. With its more ergonomic design, the SPS has the potential to decrease risks associated with intraocular injections by improving the accuracy and precision of the injection.

Development of a specific cytolethal distending toxin (cdt) gene (Eacdt)–based PCR assay for the detection of Escherichia albertii

Many Escherichia albertii isolates, an emerging pathogen of human and birds, might have been misidentified due to the difficulty of differentiating this bacterium from Escherichia coli and Shigella spp. by routine biochemical tests, resulting in underestimation of E. albertii infections. We have developed a polymerase chain reaction (PCR) assay that targets E. albertii cytolethal distending toxin (Eacdt) genes, which include the genes previously identified as Escherichia coli cdt-II. This assay could generate a single 449-bp PCR product in each of 67 confirmed E. albertii strains but failed to produce PCR product from any of the tested non–E. albertii enteric strains belonging to 37 different species, indicating 100% sensitivity and specificity of the PCR assay. The detection limit was 10 CFU per PCR tube and could detect 105 CFU E. albertii per gram of spiked healthy human stool. The Eacdt gene-based PCR could be useful for simple, rapid, and accurate detection and identification of E. albertii.

Feasibility of single-cell analysis of model cancer and foetal cells in blood after isolation by cell picking.

The objective of the present feasibility study was to transfer single cell line cells to either microscopy slides for downstream immune characterization or to polymerase chain reaction tubes for downstream DNA quantitation. Tumour cell lines, SKBR3 and MCF7 and trophoblast cell line JEG-3 were spiked in healthy donor blood. The CytoTrack system was used to scan the spiked blood samples to identify target cells. Individual target cells were identified, picked by use of a CytoPicker and deposited to either a microscopic slide or a polymerase chain reaction tube (PCR). Single tumour cells on microscopic slides were further immunostained with human epidermal growth factor receptor 2 (Her2) and epithelial cell adhesion molecule (EpCAM). From the picked cells in polymerase chain reaction tubes, DNA was amplified, quantified and used for Short Tandem Repeat genotyping. Depositing rare cells to microscopy slides was laborious with only five cells per hour. In this study with a trained operator, the picked cells had an 80.5% recovery rate. Depositing single trophoblast cells in PCR tubes was a faster process with 10 cells in 5 min. Immunostaining of isolated cells by both Her2 and EpCAM was possible but showed varying staining intensity. Presence of trophoblasts and contaminating white blood cells in PCR tubes after cell picking was confirmed based on DNA yield and mixed Short Tandem Repeat profiles in five out of eight samples. Using the CytoPicker tool, single tumour and trophoblast cells were successfully isolated and moved from blood samples, allowing subsequent immunostaining or Short Tandem Repeat genotyping.

Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood.

A molecular diagnostic method for robust detection of Ebola virus (EBOV) at the point of care (POC) directly from blood samples is described. This assay is based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) of the glycoprotein gene of EBOV. Complete reaction formulations were lyophilized in 0.2-mL polymerase chain reaction tubes. RT-LAMP reactions were performed on a battery-operated isothermal instrument. Limit of detection of this RT-LAMP assay was 2.8 × 102 plaque-forming units (PFU)/test and 1 × 103 PFU/test within 40 minutes for EBOV-Kikwit and EBOV-Makona, respectively. This assay was found to be specific for the detection of EBOV, as no nonspecific amplification was detected in blood samples spiked with closely related viruses and other pathogens. These results showed that this diagnostic test can be used at the point of care for rapid and specific detection of EBOV directly from blood with high sensitivity within 40 minutes.

Handheld energy-efficient magneto-optical real-time quantitative PCR device for target DNA enrichment and quantification

A PCR provides not only valuable genetic information that enables precise differential disease diagnosis but also quantitative data to assess various clinical states. We report the successful integration of novel dual-mode magnetic Fe3O4 nanoclusters that deliver photothermal conversion. The clusters can be excited with pulsed laser light for precision thermal cycle modulation to develop an ultrafast quantitative PCR system. Traditional PCR heats and cools the sample from outside the PCR tube; the heat needs to pass through the heat block, tube wall and transfer to the DNA through water molecules. In contrast, our system uses nanoparticles inside the liquid phase as numerous ‘nanoheaters’; thus the thermal transfer between particles and adjacent water or DNA molecules becomes extremely efficient because of proximity at the molecular level. Moreover, the defective mitochondrial DNA from cybrid cell lines of a patient with chronic progressive external ophthalmoplegia syndrome, a mitochondrial disease, was efficaciously detected. The system has a simple design, is extremely energy efficient and is faster than traditional qPCR. Our finding provides new insight into rapid and accurate quantitative diagnostics for future point-of-care applications. Iron nanoparticles play double duty as ‘nanoheaters’ for DNA amplification and as molecular manipulators in a new point-of-care prototype. Polymerase chain reaction (PCR) devices create multiple copies of small DNA strands for genetic testing, but usually require bulky heating and cooling mechanisms. Dar-Bin Shieh and colleagues from National Cheng Kung University in Taiwan have found that tiny clusters of iron oxide nanocubes can improve heat transfer in PCR devices by entering the liquid phase next to the target DNA. When irradiated by laser light, the iron nanoclusters release heat to precisely modulate DNA temperatures. Furthermore, functionalizing the nanoparticles with nucleotide probes enables them to attach to and extract specific disease-indicating mitochondria using magnetic fields. The portable, battery-powered prototype can wirelessly interface with tablet computers and is twice as efficient at isolating mitochondria as commercial kits. A novel dual-mode magnetic Fe3O4 nanoclusters was developed that exhibit efficient photothermal conversion and superparamagnetism. The particles served as numerous ‘nanoheaters’ in a PCR reaction mixture while enabling effective magnetic-based macromolecular manipulation. The thermal transfer between particles and adjacent water and DNA molecules thus become extremely efficient because of molecular-level proximity and allowed rapid amplification cycle progression. The defective mitochondrial DNA from cybrid cell lines of a patient with chronic progressive external ophthalmoplegia syndrome was efficaciously quantified by fluorescence intensity detection. Our finding provides new insight into rapid and accurate quantitative DNA diagnostics for future point-of-care applications.

Abstract P1-02-01: Genomic analysis of single cells isolated by a pulse laser retrieval system

Abstract Background: Isolating tumor cells of interest and harvesting histologically pure samples is important for genomic studies. Laser capture microdissection (LCM) is an established method to obtain such purified cell populations for various applications including DNA, gene expression, and single cell analyses. However, LCM possesses problems such as limited optical resolution, cell fragmentation from dissection, and adherence of adjacent tissue to the cells which interrupts with single cell isolation from tissue sections. To overcome these obstacles, we developed a high-throughput pulse laser retrieval system which uses a wavelength that minimizes damage to the cellular content and is processed with a sacrificial layer that provides applicable optical resolution. The aim of this study was to evaluate the performance of the pulse laser retrieval system to provide appropriate samples for genomic analysis using breast cancer tissue. Methods: An indium tin oxide (ITO) coated glass slide was prepared using fresh frozen breast cancer tissue sections of 4㎛ thickness and stained by hematoxylin and eosin. The slide was mounted on the cell isolation machine and imaging was performed with a charge-coupled device camera using a 20× lens. Following identification of the target cells by a pathologist, nano-second pulsed laser (wavelength= 1064nm) was irradiated on the target. Isolated cells were collected in a polymerase chain reaction tube and whole genome amplification (WGA) was carried out using Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Amplified genomic DNA was fragmented and Illumina sequencing libraries were constructed. Sequencing was carried out to generate data with 0.1∼0.2× depth throughout the whole genome for each sample. Copy number variation (CNV) was analyzed by the Variable binning algorithm. Results: Whole genome amplification was performed using bulk tissue and 10 captured single cells from the same specimen. No difference in amplification coverage was observed between the two samples. A CNV analysis of captured single cells revealed similar CNV profiles with those in a matched bulk tumor. Whole exome sequencing (WES) of captured single cells yielded a variant frequency of 15% at a read depth of 15× and 50M base coverage, compared to 0% at 100× and 50M for WES using bulk tumor and 0.5% at 1200× and 100K for targeted sequencing using bulk tumor. Laser capture was performed for DCIS and stromal cells from the same slide. CNV analysis of the two samples showed minimal CNV in normal stromal cells in contrast to DCIS where multiple CNVs were observed. Conclusions: Newly developed pulse laser retrieval system is suitable for capturing single cells for genomic analysis of breast cancer. WGA, WES, and CNV analysis was successfully carried out using the captured single cells and showed no difference in profile compared to those performed with bulk tissue. This method may have the potential to replace LCM for certain applications such as single cell analyses. Citation Format: Lee H-B, Kim S, Lee K-M, Jung Y, Lee AC, Kim J, Bae S, Ryu HS, Yoo T-K, Moon H-G, Noh D-Y, Kwon S, Han W. Genomic analysis of single cells isolated by a pulse laser retrieval system. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-02-01.

Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment

We have developed a method for marine diatom transformation by microparticle bombardment using polymerase chain reaction (PCR)-amplified DNA fragments. We constructed a circular vector (approximately 5000bp) containing an fcpA promoter from Phaeodactylum tricornutum, antibiotic-resistance genes and terminator from Cylindrotheca fusiformis (a “gene cassette”). Then the various lengths of linear vectors (+0–+1000 linear vectors) were then PCR-amplified from the circular plasmid. The transformants of P. tricornutum transfected with the linear vectors were obtained in the triplicate experiments. Transformation efficiencies using PCR-amplified short linear vectors containing the gene cassette and additional DNA regions of 0, 50, and 500bp at both ends of the gene cassette (+0–+500 linear vectors) did not significantly differ from one another or from the efficiency of the +1000 linear vector. Transformation efficiencies using the linear vectors were lower than that using the circular vector, but were not significantly different. The ratios of the number of transformants containing the whole region of the gene cassette to those of transformants transfected using linear vectors of various lengths were determined. An extension (≧50bp) of DNA fragments was effective for introducing the whole region of the gene cassette into the genomic DNA. In using various amounts of the +50 linear vector (37.5–300fmol/shot), we observed that transformation efficiencies using 37.5fmol (52.2ng)/shot of the linear vector were not significantly different from those obtained using 300fmol of the linear vector. The 300fmol quantity was set considering the quantity of the circular plasmid (1μg=approx. 300fmol) and the 37.5fmol quantity was set for quick and easy preparation of approximately 500ng of the linear short vector needed for triplicate transformation experiments in one PCR tube containing 50μl of PCR cocktail. Integrating the gene cassette of the short linear vectors as well as that of the full length of the linear vector (+1000 linear vector) into the chromosomal DNA was determined using Southern blot analysis. The short linear vectors tended to result in smaller numbers of insertions than those of the supercoiled plasmid. This simple and time-saving transformation method using microparticle bombardment with PCR-amplified DNA fragments permitted both functional analysis of diatom-specific genes and development of diatom strains useful for further biotechnological applications.