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MINICUBE PCR A NEW TOOL FOR GENETIC RESEARCH

MINICUBE PCR A NEW TOOL FOR GENETIC RESEARCH

Publish date: 2017-11-15

The importance of research in our genes cannot be questioned. Genetic studies have led to personalized medicine strategies in which cancer in a foreseeable future can become an if not curable then manageable disease. It has led to our understanding of inheritable diseases and strategies to handle them. Another field of genetics is the mapping of infectious diseases in epidemics and the use of this information to create new cures for diseases as diverging as HIV, HPV, and anti-biotic resistant bacteria.

Recently, we had the opportunity to get involved with a cutting-edge tool for genetic research developed by the company GNAcode from Denmark. They have designed and produced the MiniCube PCR, an asynchronous thermo-cycler. This 16 well PCR machine, quite different from other thermo-cyclers, allows multiple different DNA amplification protocols or incubations to be carried out in parallel, time-shifted parallel or serial. Potentially opening for an unseen complexity in experimental design but also giving the user a flexibility in experimentation not possible with other devices.

SCIPY FOR MINICUBE BY OUR COMPANY

We come to our role in this whole process, namely: the Open-Source Scientific Python software. The machine is equipped with an Open-Source Application interface to allow the users to create their own programs that transfer protocols to the machine for execution, monitor status, and extract and analyze thermal data related to the experiments. We took upon us the task to create a Jupyter Notebook environment for communication with the machine and then setup an example experiment and then assign it to the machine for execution. Basic work – but the frame for lots of new Jupyter Notebook applications creating advanced protocols as e.g. ”precision gradient step” experiments for primer optimization, ”calorimetric analysis of evaporation”, ”thermal analysis of reactions” to name a few.

The development is Open-Source and available and we have really appreciated the opportunity to be the first-external-developers on this platform. Scientists can now connect with Jupyter Notebook to the MiniCube and create really exciting new applications for genetic experimentation and analysis. Naturally, we are available for consultancy or service if required. At the same time, we would like to say it is a real pleasure to work with the engineers and scientists of the GNAcode company.

CERTIFIED PARTNER

We really appreciate the Open-Source opportunity allowing external professionals to create biotech software on such an advanced instrumentation system for genetic research.

We are honored to announce that we have become an official certified Open-Source business developer for the Minicube Platform from GNAcode.

THE POLYMERASE CHAIN REACTION

The polymerase chain reaction abbreviated PCR is a technique where a fragment of DNA is copied using a thermo-stable bacterial enzyme called a polymerase. This technique (see notes below) using cooling and heating cycles to separate the DNA and creating a new set of copies of the fragment can be repeated - till we receive a high quantity of DNA allowing analysis of the fragment with other techniques.

ASYNCHRONOUS PCR BY THE MINICUBE

The Minicube PCR machine can as an example run a PCR protocol on e.g. 4 wells, then later start an incubation at e.g. 50 °C at two other wells running a reverse transcription where messenger RNA is converted to cDNA and then at some point later start a second incubation at one well at 40 °C, and a co-worker can insert a row of 8 tubes an initiate a second PCR reaction. To fully explain the asynchronous function of the MiniCube PCR it can run 16 different protocols at the same time meaning 16 different PCR protocols with different temperature stages.  That is possible due to the system of single good control coupled with the thermal zero-crosstalk features of the system allowing perfectly controlled thermal reactions to co-exist within just a few millimeters from each other.

The device is ideal for optimization of protocols, preparation for sequencing, cloning, multiple parallel incubations. In normal mode, working on standard equipment, these research had to be conducted in series, now everything can be run in parallel.

35 cycles in 21 minutes and 40 s are possible and all because of the peak ramping speed of 9°C/sec. Each of the MiniCubes 16 wells is NIST temperature calibrated and during runtime, the temperature is adjusted 100 times per second by a microprocessor.

NOVEL POSSIBILITIES IN PCR 

The MiniCube PCR is a real breakthrough for thermo-cycling – not due to its high flexibility which is a “very nice feature”, but due to the very high degree of both accuracy and precision of the device coupled to the control-feedback ensuring perfectly controlled PCR reactions which not really has been achieved other than in lab-on-chip systems and due to the features the control-feedback enables.  According to the inventors of the device, it can amoung many things be utilized to monitor the evaporation of liquid from the test tubes since each well effectively is a precision calorimeter which can measure the amount of mass in the tube.

MINICUBE APP FROM APPLE APP STORE

What else? - you will ask, and I would say: MiniCube App. The iPad App (search for “GNAPCR” in Apple App store) gives full control over the process, planning of experiments can be done in the office and execution later in the lab. The iPad can control multiple machines at the same time, and other users can equally at the same time have parallel access to the MiniCube or MiniCubes – making it a great tool in a busy community lab.

Notes: Polymerase Chain Reaction

The polymerase chain reaction abbreviated PCR is a technique where DNA is copied using a thermo-stable bacterial enzyme called a polymerase which can withstand boiling. The DNA consisting of two double strands is initially heated in which the thermal energy causes the two double strands to separate – in our cells this occurs at 37 °C using more complex enzymes but the PCR technique uses simply the heat to separate the DNA and the bacterial enzyme can withstand it because it is isolated from bacteria normally living in hot springs where life exists at boiling point. Now the DNA is separated and the machine cools the test-tubes down.

The reaction soup in the test-tubes contains smaller pieces of DNA called primers, typically 20 bases long, which can create a binding to the separated DNA strands at an elevated temperature typically between 50-65 °. It also among other things contains nucleotides which are the building blocks of DNA. When the temperature reaches the “binding temperature” also called “annealing temperature” of the primers they will bind to the separated DNA strands and the polymerase can use this complex of “primer” and DNA strand to initiate elongation or copying of the DNA strand by incorporation of new nucleotides from the point where the primer ends. Now, two new strands of DNA will be formed.

The PCR technique uses two different primers and they can be designed so that one primer binds to one DNA strand and the other primer binds to the opposite DNA strand and a double-stranded DNA fragment is formed which is the length of the distance between the two primers including themselves. If the reaction is heated again the newly formed double-stranded DNA will be melted again forming a double number of strands equal to 2x where x is the number of times the heating and cooling cycle is repeated. Typically, PCR reactions are carried out in a range of heating and cooling cycles between 30-40 times. This gives a copy number of 240 equal to 1099511627776 number of DNA strands made out of a starting material of just 1 DNA copy.

In reality, this number is never so high because there is a limitation to how many copies can be built due to how many nucleotides and primers that initially are in the reaction mixture.

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