Nisha V. Padhye

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

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.

Ranque-hilsch vortex tube thermocycler for DNA amplification

Abstract 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%.

Genotyping of DNA using sequence-specific methyltransferases followed by immunochemical detection.

Abstract Modern molecular genetics relies on the ability to map the positions of genes on chromosomes, relative to known DNA markers. The first such DNA markers described were Restriction Fragment Length Polymorphisms, but any restriction endonuclease used for RFLP mapping is just one member of a restriction-modification pair. For each restriction endonuclease, there is a companion methyltransferase (MTase) that has the same DNA sequence specificity. Therefore, in principle, it should be possible to use MTases rather than restriction enzymes to detect polymorphic sites in DNA. We have used sequence-specific DNA MTases to detect genetic polymorphisms in closely related viral pathogens. If at least one MTase recognition site is present in PCR-amplified DNA, then methyl groups are incorporated; if no MTase site is present, then methyl groups are not incorporated. When several different sequence-specific DNA MTase reactions are carried out, the pattern of methyl incorporation defines a DNA MTase genotype. DNA MTase Genotyping (DMG) can be used to rapidly diagnose heritable or infectious diseases, to immunochemically detect DNA at defined 2 to 8 base pair sites, or to characterize the amplicons by constructing ordered maps.

Thermal analysis of the vortex tube based thermocycler for fast DNA amplification: Experimental and two-dimensional numerical results

In this article, experimental and numerical analyses to investigate the thermal control of an innovative vortex tube based polymerase chain reaction (VT-PCR) thermocycler are described. VT-PCR is capable of rapid DNA amplification and real-time optical detection. The device rapidly cycles six 20μl 96bp λ-DNA samples between the PCR stages (denaturation, annealing, and elongation) for 30cycles in approximately 6min. Two-dimensional numerical simulations have been carried out using computational fluid dynamics (CFD) software FLUENT v.6.2.16. Experiments and CFD simulations have been carried out to measure/predict the temperature variation between the samples and within each sample. Heat transfer rate (primarily dictated by the temperature differences between the samples and the external air heating or cooling them) governs the temperature distribution between and within the samples. Temperature variation between and within the samples during the denaturation stage has been quite uniform (maximum variation a...

CFD analysis of thermal control in a vortex tube based polymerase chain reaction chamber

In this paper, numerical analys is to investigate the thermal control of an innovative vortex tube based polymerase chain reaction thermocycler (VT -PCR) is described. VT PCR is capable of rapid DNA amplification and real -time optical detection. The device rapidly cycles six 20 µL 96 bp �-DNA samples between the PCR stages (denaturation, annealing and elongation) in approximately 6 mi nutes. Two -dimensional numerical simulations have been carried out using CFD software FLUENT v.6.2.16. Heat transfer rate (primarily dictated by the temperature differences between the samples and the external air heating or cooling them) governs the tempe rature distribution between and within the samples. Temperature variation between and within the samples during the denaturation stage has been quite uniform (maximum variation around ±0.5 uC and 1.6uC, respectively). During cooling, by adjusting the cold r elease valves in the VT -PCR during some stage of cooling, the heat transfer rate has been controlled. Improved thermal control, which increases the efficiency of the PCR process, has been obtained by slightly decreasing the rate of cooling. Thus, almost un iform temperature distribution between and within the samples (within 1 uC) has been attained for the annealing stage as well.

Fast and efficient generation of influenza A virus like particles from synthetic genes

Background With the recent emergence of the bird flu in many European countries, molecular biologists are challenged more then ever to advance the present methods of vaccine development. We have developed a method that is based on the following key elements: safety, efficacy and rapidity. Influenza A virus like particles (VLP) were generated in insect cells by co-transfection of especially designed plasmids. In order to produce VLPs, four recombinant baculoviruses were generated each containing two influenza genes under control of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) pH and p10 promotors for high level expression in Sf9 insect cells. VLPs contained 8 of 10 influenza A virus proteins of strain PR8, missing NS1 and NS2. Alternatively, VLPs were generated, by assembly of just three proteins, HA, NA and M1, which are responsible for induction of the immune system in vivo. All influenza genes have been produced from synthetical oligonucleotides, using a rapid thermocycler, the PCRJet®. Synthetic genes of new emerging influenza A variants can be produced accurately and rapidly by using this technology. Recombinant baculoviruses were generated using the Bac-to-Bac system by homologous recombination between a transfervector and the baculoviral shuttlevector (bacmid) in DH10Bac cells. By optimizing DNA synthesis and gene transfer into Sf9 cells, we anticipate major improvements in flexibility, speed and yield of influenza vaccine production as compared to available technologies. Results Co-transfection of bacmids resulted in the generation of influenza A virus like particles in the supernatant of Sf9 cells. VLPs were purified by means of Sucrose gradient centrifugation and the expected results were confirmed by Electron microscopy, Western Blot analysis and hemagglutination assays. from The 4th Recombinant Protein Production Meeting: a comparative view on host physiology Barcelona, Spain. 21–23 September 2006