Table of Contents
- Molecular technology has become an indispensable tool in contemporary biology. DNA analysis is a common requirement of many genetic procedures, although it can be time-consuming to perform by hand.
- An automated piece of equipment called a DNA analyzer, genome analyzer, or DNA sequencer is a good alternative to the tedious and error-prone process of examining DNA by hand.
- With a DNA analyzer, inexpensive and practical DNA fragment analysis is possible. Automating the sampling process also helps reduce the potential for contamination.
DNA Analyzer Principle
- A DNA analyzer is a piece of automated laboratory equipment that uses capillary electrophoresis to sequence and analyse DNA fragments of varying sizes.
- Here, a cathode is inserted into the sample, and an electric field is applied, causing the negatively charged DNA to migrate along the capillary towards the anode.
- Smaller DNA fragments travel more quickly than larger DNA fragments and so reach the detector more quickly.
- A fluorescently-labeled primer is linked to the DNA. After passing through the detection window, a focused beam of light from the laser excites the fluorescent dyes, revealing the DNA that was identified with the fluorescent primers.
- The light from the excitation has a longer wavelength than the laser’s. These photons will travel via the diffraction grating and into the CCD (charged-coupled device) detector.
- CCD utilises wavelength to detect DNA. The observed peaks are picked up by an internal size ladder, allelic ladder, and software, which then assigns an allele label to the corresponding locus.
- An electropherogram can be created by adding together the peaks of different fluorescent dyes.
Parts of DNA Analyzer
- Doors: The DNA analyzer has two doors: one for the oven and one for the instruments. In this instrument, the oven door is located on the interior. The device has a glass panel on the exterior.
- Power button: The DNA analyzer has a power button on the outside that allows you to activate or deactivate the device.
- LED indicators: There are LED lights located next to the power button that show whether or not the machine is on and functioning.
- Polymer pouch/reservoir: In this case, the polymer pouch or reservoir is essential, as it stores the material needed for the experiment.
- Polymer delivery pump: The polymer supply tube and the connecting tube link the pump to the polymer reservoir and the anode buffer container, respectively. It’s a polymer pump that feeds the array.
- Pump block: Pumping apparatus with syringe fittings, water seal, piston, pump chamber, and water trap. It regulates the pump responsible for delivering the polymer.
- Lower polymer block: It’s a buffer valve made out of polymer, located in the lower block. It regulates the flow of anode from the buffer container and is linked to the polymer delivery pump.
- Capillary array: Electrophoretic separation of fluorescently labelled DNA fragments is made possible by a capillary array, which may be replaced if necessary.
- Heat plate: The capillary array in your lab can stay at a consistent temperature with the aid of a heat plate.
- Autosampler: Cathode buffer reservoir and sampling plate are stored in the autosampler. It aids in automatically lining up the container with the cathode buffer reservoir.
- Cathode Buffer reservoir: The 1x running buffer in the cathode buffer reservoir is used to facilitate electrophoresis and keep the fluid level stable during the experiment.
- Waste reservoir: After an experiment is done, any leftover materials, such as buffers, water, or polymers, can be stored in a reservoir until disposal.
- Water reservoir: Storage tank for liquids; supplies usable in scientific procedures.
- Computer software: The software used to operate a DNA sampler varies in terms of kind, version, and functionality depending on the firm that makes the device.
- 96-well plate: This plate is very like the ELISA kit.
Operating DNA Analyzer
Calibration and correct setup are crucial for using a DNA analyzer effectively. The DNA analyzer’s calibration and initialization process consists of the following steps:
Calibrating a DNA Analyzer
The DNA analyzer undergoes both spatial and spectral calibration.
- Spatial calibration: A spatial calibration is the initial calibration of a measuring device. For this calibration, the programme must be uninstalled and reinstalled. It is also done if the instrument is moved or the capillary and plate are altered.
- Spectral calibration: When a new dye is introduced, the laser is adjusted, the CCD camera is replaced, abnormal peaks persist, or the plate capillary array is flipped, a re-calibration is required. Calibrating with a standard means passing that standard through each of the capillaries. Matrix from the various capillaries are approved or denied based on the specific spectral module of each standard. (Both the hardware and the module are included.)
Setting Up the Instrument
- Simply choose the appropriate capillary array and insert it into the device. Once you’ve got everything where it needs to be, go ahead and do some spatial calibration.
- Once you’ve decided on a polymer, you can insert the bag containing the polymer into the device. Check to see if there is enough polymer to last the entire cycle.
- Turn the autosampler to the forward position, carefully disassemble each container, and pour the contents into the water and buffer reservoir. After rinsing (first with deionized water and then buffer/water, respectively) and cleaning (with lint-free clothing), slowly add 80 ml of the buffer and water in the corresponding container. The autosampler can be moved to the appropriate location by placing the buffer in the appropriate tray, closing the instrument door, and pressing the tray button.
- For the anode buffer, unscrew the jar and rinse it off with deionized water. Use the anode buffer for a second rinsing. Fill the jar up to the 67 ml mark with anode buffer. Before re-screwing the jar into the device, check that the electrode is still submerged inside the jar.
Operating Procedure of DNA Analyzer
Following proper calibration and initialization of the DNA analyzer’s components, the following procedures must be carried out in order to complete a successful sequencing cycle:
- Start by putting the plate together properly.
- The plate must be placed within the tool.
- Simply launch the programme on the computer and set the appropriate date and time for the run.
- Play through the sequence of events. There is a wide range of possible DNA sequencing times, from 60 minutes for a normal DNA sequencing run to 120 minutes for a long read DNA sequencing run.
- During or after a sequencing run is finished, the electropherogram and run data history can be seen in real time.
Applications of DNA Analyzer
The DNA analyzer is cutting-edge technology that helps scientists do DNA analysis in a variety of settings. It may work for Sanger sequencing, genotyping, STR and SNP profiling, gene mutation analysis, etc. The DNA analyzer can be used for the following:
- Forensic science: Science of the Aftermath; Forensics Forensic scientists frequently employ molecular methods, particularly gene sequencing. To sum up, a DNA analyzer will significantly cut down on the time needed for DNA sequencing by replacing most of the labour that was previously done manually. The risk of pollution spreading will also be reduced. A DNA sampler is also useful for evaluating isolated DNA sequences.
- Facilities devoted to studying microorganisms: To study microorganisms, gene analysis of bacteria and viruses is a common method. As a result, a DNA analyzer can be a great alternative to several hours of manual labour.
- Cancer-related laboratories: For laboratories specialising in cancer research, the DNA sampler’s ability to detect genetic mutation will be invaluable in determining the disease’s underlying cause.
Advantages of DNA Analyzer
- Large quantity of sample processing: The capillary array can process between 48 and 96 samples in parallel, allowing for a greater throughput of samples.
- Less time-consuming: Time savings due to its ability to process several samples with minimal human intervention. It’s also automated, so while the machine is operating, the worker may focus on anything else.
- Faster: at least 48 samples can be analysed by DNA analyzers with the help of only skilled personnel. Consequently, it improves the effectiveness of scientific research facilities.
- Less cross-contamination: Since robotic sample processing eliminates the need for human intervention, there is less of a danger of cross-contamination.