High-throughput Polymerase Chain Reaction Research Articles

High-Throughput Polymerase Chain Reaction in Parallel Circular Loops Using Magnetic Actuation

We report here a novel multichannel closed-loop magnetically actuated microchip for high-throughput polymerase chain reaction (PCR). This is achieved by designing a series of concentric circular channels on one microchip and exploiting a magnetic force to drive DNA samples flowing continuously through the closed loops. The magnetic force arises from an external permanent magnet through ferrofluid plugs inside the microchannels. The magnet enables simultaneous actuation of DNA samples in all the channels. As the samples go around the loops, they pass through three preset temperature zones. Parameters of PCR, such as incubation time, temperatures, and number of cycles, can be fully controlled and adjusted. High reproducibility was achieved for different channels in the same run and for the same channels in consecutive runs. Genetically modified organisms (GMOs) were amplified simultaneously using the developed device. This simple, reliable, and high-throughput PCR microchip would find wide applications in forensic, clinical, and biological fields.

Development and evaluation of a sensitive enzyme-linked oligonucleotide-sorbent assay for detection of polymerase chain reaction-amplified hepatitis C virus of genotypes 1–6

A high-throughput polymerase chain reaction (PCR)-based enzyme-linked oligonucleotide-sorbent assay (ELOSA) was developed for use in the diagnostic testing of serum from patients who may be infected with different hepatitis C virus (HCV) genotypes. Twelve genotype-specific 5′-aminated DNA-coated probes were designed based on the variable 5′-untranslated region sequences of the HCV genotypes 1–6. Using 100 clinical serum samples, the performance of the PCR-ELOSA method was compared with Roche's COBAS Amplicor HCV Monitor V2.0 assay and the VERSANT HCV genotype assay (LiPA), and the overall agreement was 99% at the level of HCV genotypes with a detection range of 2.0 × 10 2 to 1.0 × 10 7 IU/ml for PCR-ELOSA. The PCR-ELOSA was more comprehensive as demonstrated by the fact that ∼20% of the samples with different subtypes could be discriminated by this method but not by LiPA. In addition, the PCR-ELOSA system showed high accuracy (CV ≤ 6.36%) and even higher reproducibility (CV ≤ 5.55%). Thus, this novel PCR-ELOSA system provides a sensitive and versatile alternative to current HCV detection assays.

Novel computational methods for increasing PCR primer design effectiveness in directed sequencing

BackgroundPolymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment.ResultsWe have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research.ConclusionThe high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.

A novel buffer system, AnyDirect, can improve polymerase chain reaction from whole blood without DNA isolation

Background Polymerase chain reaction (PCR) using DNA from blood samples is a valuable tool in the field of medical diagnostics. However, DNA isolation from blood is a laborious and sample-consuming step, and hampers the automation of PCR for large-scale studies. Attempts to perform PCR from blood without DNA isolation have been difficult to achieve, since numerous endogenous and exogenous blood constituents may inhibit PCR. Methods We used a novel buffer system, ‘AnyDirect’, that conserves the enzymatic activity of DNA polymerases for effective use in direct PCR from whole blood under various conditions. Results Using AnyDirect, DNA amplification was achieved from whole blood with a variety of thermostable DNA polymerases. Amplification occurred regardless of target size (up to 1.7 kb), presence of various known PCR inhibitors, and high target GC content. Importantly, low copy number DNA targets were effectively amplified from whole blood. Conclusions AnyDirect buffer allows direct PCR from whole blood and may facilitate detection of genetic diseases or infections by eliminating the time and effort for DNA extraction. The use of AnyDirect could facilitate the development of high-throughput PCR for large-scale diagnostic screening or investigation of various medical conditions.

A novel miniaturized PCR multi-reactor array fabricated using flip-chip bonding techniques

This paper describes a novel miniaturized multi-chamber array capable of high throughput polymerase chain reaction (PCR). The structure of the proposed device is verified by using finite element analysis (FEA) to optimize the thermal performance, and then implemented on a glass–silicon substrate using a standard MEMS process and post-processing. Thermal analysis simulation and verification of each reactor cell is equipped with integrated Pt temperature sensors and heaters at the bottom of the reaction chamber for real-time accurate temperature sensing and control. The micro-chambers are thermally separated from each other, and can be controlled independently. The multi-chip array was packaged on a printed circuit board (PCB) substrate using a conductive polymer flip-chip bonding technique, which enables effective heat dissipation and suppresses thermal crosstalk between the chambers. The designed system has successfully demonstrated a temperature fluctuation of ±0.5 °C during thermal multiplexing of up to 2 × 2 chambers, a full speed of 30 min for 30 cycle PCR, as well as the capability of controlling each chamber digitally and independently.

Novel Convective Flow Based Approaches for High-Throughput PCR Thermocycling

A critical need exists for the development of next-generation genomic analysis instrumentation capable of offering significantly higher throughput at a lower cost than current technology. In this paper, we explore the potential of natural convection-based systems to address these issues by providing a thermocycling hardware platform capable of performing rapid polymerase chain reaction (PCR) amplification of DNA. These systems can be arrayed in a multi-well format that is simple to operate, is suitable for integration with high-throughput automated liquid handling systems, and can be easily and inexpensively mass-produced.

High-throughput polymerase chain reaction chemiluminescent enzyme immunoassay for typing and quantifying human papillomavirus DNAs

A miniaturized polymerase chain reaction (PCR) chemiluminescent enzyme immunoassay (CLEIA) based on a 384-well microtiter plate for detection and typing of oncogenic high- and low-risk human papillomavirus (HPV) in genital lesions is described. The assay relies on PCR consensus amplification, hybridization of the digoxigenin-labeled product by means of type-specific biotin-labeled oligoprobes immobilized on the streptavidin-coated wells of a 384-well microtiter plate, and quantification by means of a horseradish peroxidase-labeled antidigoxigenin antibody and chemiluminescence detection. The method provides semiquantitative information on the viral load, with a limit of detection of 10–50 DNA copies for HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 54, 58, and 59 and high reproducibility (intraassay CV 7.5%, interassay CV 9.5%). Results obtained on 60 clinical samples were concordant with those obtained with a conventional PCR-enzyme-linked immunosorbent assay colorimetric assay. The 384 PCR-CLEIA method, which is amenable to automation, represents a fast and high-throughput method for detecting and typing HPV DNAs in screening programs and evaluating the viral load.

RNA labeling and hybridization of DNA microarrays.

AbstractDNA array technologies provide powerful tools for comprehensive genomic and transcriptomic exploration in renal research. Applications of DNA arrays include analysis of disease predisposition by using single-nucleotide polymorphism (SNP) microarrays (1), reliable detection of chromosomal deletions and amplifications using microarray-based comparative genomic hybridization (array-CGH) (2), and global gene-expression patterns by cDNA microarrays (3). A DNA microarray consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern. Genetic content for DNA arrays may consist of cDNA fragments amplified from extensive (EST) clone libraries by high-throughput polymerase chain reaction (PCR) methods. Production of cDNA arrays is error-prone because of requirements for maintenance and processing of large numbers (tens of thousands) of bacterial stock of expressed sequence tag (EST) clones. Synthetic long oligonucleotide probes 45-mer to 70-mer), designed for optimal sequence specificity and annealing temperatures, can be spotted as an alternative to cDNA fragments, and pose lesser logistic and quality-control demands during array production. Ready-to-print oligonucleotide libraries containing probes for human, mouse, or rat Unigene clusters are available from several vendors. Although precise knowledge of exon-specific probe sequences can be a distinct bioinformatics advantage for spotted oligonucleotides, it may pose a considerable problem for genes with extensive alternative splicing of exons.KeywordsSodium Dodecyl SulfateArray ProductionStandard Saline CitrateQIAquick Polymerase Chain Reaction PurificationSpotted cDNAThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Micro-assembled multi-chamber thermal cycler for low-cost reaction chip thermal multiplexing

Abstract This paper presents a miniaturised multi-chamber thermal cycler capable of thermal multiplexing for high throughput polymerase chain reaction (PCR) of nucleic acids, using low-cost reaction chip. The thermal cycler has been fabricated in a micro-assembly manner using flip–chip bonding technique, which is batch manufacturable with good reproducibility. Silicon heating blocks in the multi-chamber array are mounted on a printed-circuit-board (PCB) substrate, with the later attached to a metal plate heat sink. Thermal cross-talk has been minimised by using thin and low thermal conductivity PCB. The preferred reaction chip containing multiple chambers is made of low-cost plastics, while other PCR compatible materials are also possible. The preliminary experiments show that with up to 20 μl sample in the thermally formed plastic chip, a full speed of 8 min for 30-cycle PCR is achievable. Thermal cross-talk of as less as 0.2% is obtained with the very thin PCB substrate (500 μm, FR4) and the plastic chip (100 μm PET), in a standard format of multi-chamber array. A temperature fluctuation of ±0.1 °C has been achieved during thermal multiplexing of up to 16 chambers, with each chamber consuming an average heating power of no more than 1.2 W. Finite element analysis (FEA) is conducted to optimise the thermal performance of the cycler. Experiments are in well agreements with the simulations.

High-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with UV detection.

Routine genetic analysis of large numbers of individuals by polymerase chain reaction (PCR) using capillary electrophoresis is often restricted by the low throughput of standard protocols and the tedious sample preparation process. Here, we demonstrate that capillary electrophoresis with UV detection can be used in PCR-based DNA analysis starting from clinical samples without purification or complicated sample manipulation. After PCR reaction using cheek cells, blood, or HIV-1 gag DNA, the reaction mixtures were injected into a capillary array either on-line or off-line by base stacking. The use of multiplexed absorption detection and the elimination of any purification steps both before and after PCR reaction can potentially provide significant benefits compared to current methods for DNA analysis with regard to time, cost, and labor.

High-Speed Screening of Polymerase Chain Reaction Products by Capillary Electrophoresis

In an effort to develop capillary electrophoresis (CE) for high-throughput polymerase chain reaction (PCR) molecular diagnostics, a method was developed to rapidly screen small PCR products of similar molecular weights. The assay of interest required the separation of two PCR products (375 and 400 bp) in an assay of TGF-β1knockout mice to determine the genotype of neonates. Using a commercially available CE instrument, the two PCR products were separated in 12 min with a replaceable gel buffer, a 20-cm effective length DB-17 capillary, and 185 V/cm field strength. With the coinjection of a 20-bp ladder, the sizes of the PCR products were determined from the electropherogram without using a calibration plot and curve-fitting program. Faster separation was obtained using the combination of a short effective length capillary and high field strength. The two PCR products were separated in 82 s with a 7-cm effective length capillary and 556 V/cm. A 60% buffer further reduced the separation time in about a minute. This high-speed separation, with minimum postrun data processing, is highly desirable for the high-throughput screening of PCR products using a single-capillary CE system.