Nanobiotechnology: the promise and reality of new approaches to molecular recognition

Nanobiotechnology is the convergence of engineering and molecular biology that is leading to a new class of multifunctional devices and systems for biological and chemical analysis with better sensitivity and specificity and a higher rate of recognition. Nano-objects with important analytical applications include nanotubes, nanochannels, nanoparticles, nanopores and nanocapacitors. Here, we take a critical look at the subset of recent developments in this area relevant to molecular recognition. Potential benefits of using nano-objects (nanotubes, quantum dots, nanorods and nanoprisms) and nanodevices (nanocapacitors, nanopores and nanocantilevers) leading to an expanded range of label multiplexing are described along with potential applications in future diagnostics. We also speculate on further pathways in nanotechnology development and the emergence of order in this somewhat chaotic, yet promising, new field

Artificial Intelligence-Powered Search Tools and Resources in the Fight Against COVID-19.

Emerging technologies are set to play an important role in our response to the COVID-19 pandemic. This paper explores three prominent initiatives: COVID-19 focused datasets (e.g., CORD-19); Artificial intelligence-powered search tools (e.g., WellAI, SciSight); and contact tracing based on mobile communication technology. We believe that increasing awareness of these tools will be important in future research into the disease, COVID-19, and the virus, SARS-CoV-2

Molecular diagnostics: hurdles for clinical implementation

In the coming years, molecular diagnostics will continue to be of critical importance to public health worldwide. It will facilitate the detection and characterization of disease, as well as monitoring of the drug response, and will assist in the identification of genetic modifiers and disease susceptibility. A wide range of molecular-based tests is available to assess DNA variation and changes in gene expression. However, there are major hurdles to overcome before the implementation of these tests in clinical laboratories, such as which test to employ, the choice of technology and equipment, and issues such as cost-effectiveness, accuracy, reproducibility, personnel training, reimbursement by third-party payers and intellectual property. At present, PCR-based testing predominates; however, alternative technologies aimed at reducing genome complexity without PCR are anticipated to gain momentum in the coming years. Furthermore, development of integrated chip devices ('lab-on-a-chip') should allow point-of-care testing and facilitate genetic readouts from single cells and molecules. Together with proteomic-based testing, these advances will improve molecular diagnostic testing and will present additional challenges for implementing such testing in health care settings

DOP-PCR amplification of whole genomic DNA and microchip-based capillary electrophoresis.

Universal or whole genome amplification by polymerase chain reaction (PCR) is a rapid and efficient method to generate fragments representing the target sequence, as well as to increase a limited amount of template. One of the most common PCR protocols for total genome amplification is the interspersed repetitive sequence-PCR (IRS-PCR) in which primers specific for human repeat-rich regions are used to generate PCR products between adjacent repeated sequences (1). However, although IRS-PCR across regions such as Alu families of human repeat has been demonstrated to be useful, the nonuniform distribution of repeat-rich region within the human genome has been a limitation. Alternative strategies have been proposed. In the primer-extension preamplification (PEP), multiple rounds of extensions with Taq DNA polymerase and a random mixture of 15-base oligonucleotides as primers produce multiple copies of the template present in the sample (2–5). In a more demanding protocol, called linker adaptor-PCR, RsaI restricted genomic DNA fragments are ligated to SmaI-cut pUC plasmid. Subsequently, the inserts are amplified by PCR using the universal M13/pUC sequencing and reverse sequencing primers and then released by EcoRI digestion (6). The tagged random primer PCR (T-PCR) is a two-step PCR strategy which consists of a pool of all possible 3'-sequences for binding to the target DNA and a constant 5'-region for the detection of incorporated primers (7). Recently, degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) was developed to allow random amplification of DNA from any source (8–10). DOP-PCR uses a partially degenerate sequence in a PCR protocol with two different annealing temperatures. It has been successfully applied for amplifying entire genomes such as human, mouse, and fruit fly, as well as isolated human chromosomes and cosmids (11). The technique has also been used to prepare whole chromosome paint probes (11,12) for micro-FISH assays (13–15), comparative genomic hybridization (16), to increase the amount of sample for genotyping (17), and genomic fingerprinting (18). The DOP-PCR primer consists of three regions. The 5'-end carries a recognition sequence for XhoI (C•TCGAG), a restriction endonuclease that cuts rarely within the human genome. This sequence can be used for cloning, if desired. The sequence is then followed by a middle portion containing six nucleotides of degenerate sequence (NNNNNN, where N = A, C, G, or T in approximately equal proportions) and a 3'-end sequence containing six specific bases (ATGTGG) which primes the reaction approximately every 4 kb (8,9). The principle of the technique is that at a sufficiently low annealing temperature only the six specific nucleotides included in the 3'-end of the degenerate oligonucleotide will anneal to the genomic strand allowing the primer to initiate PCR. The PCR fragments are then generated which contain the full length of the oligoprimer at one end and its complementary sequence at the other end. Subsequently, the temperature is increased to the level required for the full length of the degenerate primer to anneal. For additional details, we direct the reader to the original papers (8,9). We have adapted the DOP-PCR technique to a three-microchip format (19). DOP-PCR amplified genomic DNA produced in a first silicon-glass chip is transferred to a second chip for a locus-specific, multiplex PCR of the dystrophin gene exons in order to detect deletions causing Duchenne/Becker muscular dystrophy (DMD/BMD). Amplicons from the multiplex-PCR are then analyzed by electrophoresis in a third microchip. The analytical performance of the microchip capillary electrophoresis (MCE) is also compared to conventional capillary electrophoresis (CE)

Surface effects on PCR reactions in multichip microfluidic platforms

We evaluated the compatibility of several common plastics, commercially available plastic tubing and disposable syringes which might be useful in the construction of microfluidic platforms with respect to the polymerase chain reaction (PCR). A simple and inexpensive plastic test module was constructed in order to evaluate some of the construction plastics. We also investigated the effect of addition of PEG 8000 to PCR reaction mixtures on the compatibility of materials. These studies identified several common plastics, plastic tubing, and disposable syringes which were compatible with the PCR reaction

Micropillar array chip for integrated white blood cell isolation and PCR

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 mm in cross-section, etched to a depth of 80 mm with adjacent pillars being separated by 3.5 mm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare TM microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip