PCR reaction consists of three temperature dependent steps: denaturation at 92-95°C, annealing at 37-65°C, and extension at 72°C. The target DNA fragment is duplicated with the completion of one cycle of the three steps. After 20 to 40 repeated cycles, the target DNA fragment is exponentially amplified and can be readily detected. The PCR reaction is commonly carried out in a thermocycler, which repeatedly performs the heating and cooling of the reaction tube to achieve the required temperature for each step of PCR. However, conventional thermocyclers are typically equipped with a hot-plate design with a metal block of high heat conductivity combining with plastic multi-well plates of low thermal conductivity, and therefore severely hinder the achievable heating and cooling rate. Consequently, the majority of time is wasted on controlling the temperature changes of the instrument, rather than driving the PCR reaction.
POCKIT™, on the other hand, is based on the technology of insulated isothermal PCR (iiPCR), which utilizes the phenomenon of natural thermal convection to drive the PCR reaction. The iiPCR reaction is done in a specially designed capillary tube, R-tube, inside the chamber of POCKIT™ which is a convective PCR device providing insulation with a single isothermal heat source. When the isothermal heating of 95°C is applied to the bottom of the R-tube, the hotter solution becomes lighter to generate a current to go up, and the cooler solution at the top is heavier to go down. Consequently, a convective current with thermal gradient along the R-tube is generated for PCR reaction: the denaturing step at the bottom, the annealing at the top, and the elongation at the middle of the R-tube. This continuous convective PCR reaction is very efficient and can decrease the regular PCR reaction time significantly without affecting the sensitivity.
Since only a single heat source is needed without complicated parts to control the temperature changes like conventional thermocyclers, the design of POCKIT™ is simple and light-weighted. In addition, since PCR is driven by the temperature gradient from the natural thermal convection without repeatedly raising and lowering the temperature, it only takes about 15-20 seconds to complete one PCR cycle, which is much more efficient than conventional PCR and can keep the reaction time short with POCKIT™.
The reagents used in POCKIT™ platform are similar to those used in conventional PCR reaction, including primers, dNTP, reaction buffers, DNA polymerases, and templates. To increase the reaction specificity, POCKIT™ is equipped with two fluorescent channels to detect the fluorescent signals of target nucleic acid. Therefore, like real-time PCR system, fluorogenic probe is also required.
The unique formulation of Uni-ii Buffer combines necessary reagents to optimize the thermal convection rate, stabilize the temperature gradient, reduce the interaction between the reaction mix and R-tube, and increase the efficiency of DNA polymerase for successful iiPCR reaction. Users can choose either high-salt or low-salt Uni-ii Buffer to optimize their primer design and develop their own detection system.
Yes, POCKIT™ can The built-in program of POCKIT™ is specially designed for both PCR and the reverse transcription (RT)-PCR reaction, and therefore both DNA and RNA template can be applied. When the reaction starts, it is first kept at 50°C for 10 minutes for RT reaction, followed by the PCR reaction. The Uni-ii Buffer is optimized to work for both PCR and RT-PCR reactions. Users simply need to add the reverse transcriptase (e.g. MMLV or AMV reverse transcriptase) for the reaction. The amount of reverse transcriptase used is the same as conventional PCR.
POCKIT™ is used to detect nucleic acid (DNA and RNA). Therefore, it can effectively detect the nucleic acid from pathogens of infectious diseases including viruses, bacteria and parasites, or mutated genes that cause hereditary diseases. However, POCKIT™ cannot be used to detect certain metabolic indicators such as blood glucose and electrolytes, non-nucleic acid pathogens (e.g. Mad Cow Disease), or chemicals/drugs.
POCKIT™ is specially designed to be used for field diagnostics. POCKIT Micro series nucleic acid analyzer is light-weighted and hand-held with rechargeable battery. POCKIT™ Xpress comes in as a carry-on hard-shell suitcase package including POCKIT™ nucleic acid analyzer, a mini-centrifuge and two micro pipettes. The specific reagent kits for POCKIT™ are lyophilized for room temperature shipping. Therefore, POCKIT™ is a portable platform and can take on any tasks at the point of need.
The limitations of the operation environment of POCKIT™ are as follows:
￭ It needs to be operated on a flat surface.
￭ The optimal operation temperature is 15-35°C.
Yes, POCKIT™ is equipped with two optical channels (520nm, 550nm) for multiplex detection. Users can detect two targets in a single reaction by labeling specific probes with unique fluorescent dyes to generate different fluorescent colors for each target (e.g. FAM at 520 nm and JOE at 550 nm).
Because POCKIT™ uses natural thermal convection to drive PCR reaction with the temperature gradient, which is different from conventional PCR, special attention is required to avoid the dimer formation between primer and probe, as well as the secondary structure within the primer/probe to minimize possible interference of reactions.
Because the emission light at 520 nm may interfere with that at 550 nm, we recommend designing the primary target at 520 nm and secondary target such as intenal control at 550 nm.
The only parameter that users can set up is the wavelength selection (520 nm, 550 nm, and 520 + 550 nm).
POCKIT™ has a built-in android smartphone chip to collect, calculate, and monitor the process of optical system before and after the reaction, transform the fluorescence change into "+" or "-" result, and directly display on the touch-panel screen. Therefore, further analysis is not needed after the reaction is completed. All information including the pictures showing the fluorescence before and after the reaction, raw fluorescent data, the ratio of the fluorescence before and after reaction, and the qualitative results is automatically saved in a SD card for users to review.
POCKIT™ is a qualitative system. However, the optical system of POCKIT™ is very similar to the one used in real-time PCR, and has the potential to be developed into a quantitative system.
Yes, but we do not recommend. In order to optimize the thermal convection rate, stabilize the temperature gradient, reduce the interaction between the reaction mix and R-tube, and increase the efficiency of DNA polymerase for successful iiPCR reaction, Uni-ii Buffer contains certain unique reagents which are not provided in other commercially available buffers. Therefore, we recommend to use Uni-ii Buffer in order to achieve optimal iiPCR reaction.
For Uni-ii Buffer, users can use commercially available enzymes and optimize accordingly. Users can start with the recommended amount used for conventional and RT-PCR, and fine-tune further based on the optimization results.
The POCKIT™ primer design rules are similar to real-time PCR, including:
￭ The amplicon length should be less than 150 bps, and the shorter the better.
￭ The melting temperature (Tm) should be in the range of 58-62°C.
￭ Avoid 4 and above repeated bases.
￭ Avoid 3 and above G/C residue of the last 5 bases at 3’-end.
￭ GC content of the primer should be 20-80%.
￭ Primer length should be 15-25 bps.
The recommended fluorogenic probe for POCKIT™ is TaqMan probe. The probe design rules are as follows:
￭ At 5’-end, the probe should be labeled with FAM dye (520 nm) or JOE / VIC dye (550 nm).
￭ The quencher dye at 3’-end of the probe should be black hole quencher (BHQ1) or minor groove binder (MGB).
￭ Tm should be in the range of 68-72°C, and should be 10°C higher than the Tm of the primer.
￭ The distance between the primer and the probe should be at least one base apart to prevent steric hindrance.
￭ Avoid 4 and above repeated bases.
The differences between the fluorogenic probe design of POCKIT™ and real-time PCR are as follows:
￭ For POCKIT™ fluorogenic probe:
(1) GC content should be 40-80%
(2) The length of the probe should be 15-30 bases, the shorter the better.
(3) The annealing between the 5’-end of the probe and the template should be strong enough with more negative Gibbs free energy.
(4) Avoid the target region of the template that tends to form secondary structures.
A successful PCR reaction is dependent on the quality of primers. The factors that may affect the primers include:
￭ Primer design, including Tm, formation of secondary structure and dimmers
￭ Primer quality, including the raw materials used to synthesize primers and purification methods.
￭ Primer concentration.
Users can design primers according to the guideline described in Q16, and also apply the following:
￭ In addition to the original design, users can synthesize more primers with the addition of one or two residues at either end to select the most optimal design.
￭ When synthesizing primers, PAGE purification is required.
￭ Order primers from more than one oligo suppliers to compare performance.
￭ Test different ratio and/or concentration of forward and reverse primers, and/or different enzyme concentrations.
￭ Use serial diluted positive samples to determine the analytical sensitivity and detection end point. The more sensitive, the better.
￭ Use PAGE analysis to determine the specificity of primer pairs. The less non-specific products on PAGE, the better.
￭ In general, the sensitivity of POCKIT™ should reach at least 100 copies per reaction.