Gene synthesis demystified.

The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mono-nucleotides. The 5′-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [γ-32P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent head-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor transfer RNA gene. The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonu-clease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene.

Total synthesis of a gene

Bacteriophages (phages) are the most abundant and widely distributed organisms on Earth, constituting a virtually unlimited resource to explore the development of biomedical therapies. The therapeutic use of phages to treat bacterial infections ("phage therapy") was conceived by Felix d'Herelle nearly a century ago. However, its power has been realized only recently, largely due to the emergence of multi-antibiotic resistant bacterial pathogens. Progress in technologies, such as high-throughput sequencing, genome editing, and synthetic biology, further opened doors to explore this vast treasure trove. Here, we review some of the emerging themes on the use of phages against infectious diseases. In addition to phage therapy, phages have also been developed as vaccine platforms to deliver antigens as part of virus-like nanoparticles that can stimulate immune responses and prevent pathogen infections. Phage engineering promises to generate phage variants with unique properties for prophylactic and therapeutic applications. These approaches have created momentum to accelerate basic as well as translational phage research and potential development of therapeutics in the near future.

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Genetic Engineering of Bacteriophages Against Infectious Diseases

Immobilization of cells inside microfluidic devices is a promising approach for enabling studies related to drug screening and cell biology. Despite extensive studies in using grooved substrates for immobilizing cells inside channels, a systematic study of the effects of various parameters that influence cell docking and retention within grooved substrates has not been performed. We demonstrate using computational simulations that the fluid dynamic environment within microgrooves significantly varies with groove width, generating microcirculation areas in smaller microgrooves. Wall shear stress simulation predicted that shear stresses were in the opposite direction in smaller grooves (25 and 50 microm wide) in comparison to those in wider grooves (75 and 100 microm wide). To validate the simulations, cells were seeded within microfluidic devices, where microgrooves of different widths were aligned perpendicularly to the direction of the flow. Experimental results showed that, as predicted, the inversion of the local direction of shear stress within the smaller grooves resulted in alignment of cells on two opposite sides of the grooves under the same flow conditions. Also, the amplitude of shear stress within microgrooved channels significantly influenced cell retainment in the channels. Therefore, our studies suggest that microscale shear stresses greatly influence cellular docking, immobilization, and retention in fluidic systems and should be considered for the design of cell-based microdevices.

/pdf/microcirculation-within-grooved-substrates-regulates-cell-4l0c26zsdi.pdf

Microcirculation within grooved substrates regulates cell positioning and cell docking inside microfluidic channels

Present and future of rapid and/or high-throughput methods for nucleic acid testing.

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1002&context=cbmesubramanian

Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes

Processes and apparatus for performing fast, precise, and reliable gas jet thermocycling on a sample using of pressurized gases delivered to a thermostated reaction chamber at high velocity using computer controlled electronic multiport valves are disclosed. Apparatus for achieving these processes are also disclosed.

Gas jet process and apparatus for high-speed amplification of dna

An innovative polymerase chain reaction (PCR) thermocycler capable of performing real-time optical detection is described below. This device utilizes the Ranque–Hilsch vortex tube in a system to efficiently and rapidly cycle three 20 μL samples between the denaturation, annealing, and elongation temperatures. The reaction progress is displayed real-time by measuring the size of a fluorescent signal emitted by SYBR green/double-stranded DNA complexes. This device can produce significant reaction yields with very small amounts of initial DNA, for example, it can amplify 0.25 fg (∼5 copies) of a 96 bp bacteriophage λ-DNA fragment 2.7×1011-fold by performing 45 cycles in less than 12 min. The optical threshold (150% of the baseline intensity) was passed 8 min into the reaction at cycle 34. Besides direct applications, the speed and sensitivity of this device enables it to be used as a scientific instrument for basic studies such as PCR assembly and polymerase kinetics.

/pdf/ranque-hilsch-vortex-tube-thermocycler-for-fast-dna-3g96k5c5g9.pdf

Ranque-Hilsch vortex tube thermocycler for fast DNA amplification and real-time optical detection

A thermal cycling apparatus (10) utilizes hot and cold gas streams produced from pressurized gas being passed through a Ranque-Hilsch Vortex Tube (20) to efficiently and rapidly cycle samples (70) (i.e., DNA+Primer+Polymerse) between the denaturation, annealing, and elongation temperatures of the PCR process. The samples (70) are disposed within a reaction chamber (40) that, through connection with a vortex tube (20), allows the gas to contact the samples (70). The temperature of the gas that is allowed to contact the samples (70) is controlled by a valving system (30) being connected with the vortex tube (20) and the reaction chamber (40). The valving system (30) controls the flow of cold gas into the reaction chamber (40) where it is mixed with the hot gas to establish the different temperatures required for the denaturation, annealing, and elongation steps of the cycle.

Vortex tube thermocycler

An innovative polymerase chain reaction (PCR) thermocycler using pressurized gas through a Ranque–Hilsch vortex tube is described below. This device can amplify 10 pg of 186 bp Escherichia coli uidA amplicon in a 20 µL sample 3.3 × 108‐fold, by performing 35 cycles in less than 8 min. This PCR amplification corresponds to an overall efficiency of 75%.

Nisha V. Padhye

Papers

Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes

Gas jet process and apparatus for high-speed amplification of dna

Ranque-Hilsch vortex tube thermocycler for fast DNA amplification and real-time optical detection

Vortex tube thermocycler

Ranque–Hilsch Vortex Tube Thermocycler for DNA Amplification