Molecular technology is one of the most prominently used techniques in modern biology. Many genetic techniques constantly require DNA analysis, so its analysis is tedious when done manually.
DNA analyzer, genome analyzer, genetic analyzer or DNA sequencer is an automated equipment that can be a suitable replacement to the manual work of analyzing DNA.
DNA fragment analysis becomes very affordable and feasible with a DNA analyzer. It will also decrease the risk of contamination as sampling handling is automated.
Table of Contents
Working Principle of DNA Analyzer
DNA analyzer is an automatic instrument where sequencing and analyzing DNA fragments is done by capillary electrophoresis based on their sizes. Here, for capillary electrophoresis, a cathode is placed into the sample, and under an electric field, the negatively charged DNA migrates to the anode through the capillary. This migration helps to separate DNA fragments; here, smaller DNA fragments migrate faster than the larger DNA fragments and reach the detector soon.
The DNA has a fluorescent-labeled primer attached. When DNA with fluorescent-labeled primers passes the detection window during migration, a narrow beam of light from the laser excites the fluorescent dyes. The excitation emits a wavelength of light longer than the laser’s wavelength. These light rays will pass through the diffraction grating, which directs them into the CCD (charged-coupled device) detector.
CCD detects DNA based on wavelength. An internal size ladder, allelic ladder, and a software program pick up the detected peaks and provides an allele designation for a given locus. This combination of all the fluorescent dye peaks gives rise to an electropherogram.
An electropherogram is a plot containing results from different analyses after the automated electrophoresis sequencing technique.
Parts of DNA Analyzer
- Door: There are two doors in the DNA analyzer; the oven door and the instrument door. The oven door is on the inner part of the instrument. The outer part of the instrument has a door with glass.
- Power button: It is present in the outermost part of the DNA analyzer, and it helps turn the equipment on or shut it down.
- LED indicators: These indicate if the machine is running or not and are present just beside the power button.
- Polymer pouch/reservoir: It provides the desired polymer for the experiment.
- Polymer delivery pump: It is connected to the polymer reservoir and anode buffer container via a polymer supply tube and interconnected tube, respectively. It pumps polymer into the array.
- Pump block: It consists of a syringe fitting array, water seal, piston, pump chamber, and water trap. Its function is to control the polymer delivery pump.
- Lower polymer block: It consists of a buffer valve. It is connected to the anode buffer container and polymer delivery pump and functions as a controller of the anode flow from the container.
- Capillary array: It is a replacement unit (8 or 24 capillaries available) and enables the separation of the fluorescent-labeled DNA fragments through electrophoresis.
- Heat plate: It helps in maintaining constant capillary array temperature.
- Autosampler: It holds the cathode buffer reservoir and sampling plate. It helps appropriately align the container and the cathode buffer reservoir without human intervention.
- Cathode Buffer reservoir: It consists of 1✕ running buffer for supporting electrophoresis and helps maintain constant fluid height during the experiment.
- Waste reservoir: It collects discarded waste like buffers, water, and polymers after the completion of the experiment.
- Water reservoir: It holds water for experimenting.
- Computer software: The type, version, and operation of the computer software depending on the company manufacturing the DNA sampler.
- 96 well plate: The 96 well plate is similar to the ELISA test kit.
Operating Genetic Analyzer
For operating a DNA analyzer, calibration and proper setup are essential. Following are the steps for calibrating and setting up a DNA analyzer:
Calibrating a DNA Analyzer
Two types of calibration are done in DNA analyzer; spatial and spectral.
- Spatial calibration: It is the calibration performed when the instrument is used for the first time. Here the software is uninstalled and reinstalled for calibration. It is also done when changes are made in the capillary and plate and when the instrument is moved.
- Spectral calibration: It is performed whenever a new dye is used, the laser is realigned, the CCD camera is replaced, incorrect peaks are seen consistently, and when plate capillary array is switched. Run calibration standard through each capillary. Each standard has a particular spectral module based on which matrices of the different capillaries are accepted and rejected. (The module is provided along with the equipment)
Setting Up the Instrument
- Select the desired capillary array and place the capillary array inside the instrument. Perform spatial calibration after proper placement.
- Then select the desired polymer and place the polymer pouch in the instrument. Make sure the amount of polymer is enough for the complete cycle.
- To fill the water and buffer reservoir, bring the autosampler to the forward position, slowly dissemble each container and discard any remaining fluid. Slowly fill 80 ml of the buffer and water in the respective container after thorough rinsing (first with deionized water and then buffer/water, respectively) and cleaning using lint-free clothes. Place the buffer in the respective tray, close the instrument door and push the tray button to move the autosampler to the correct position.
- Filling anode buffer jar: Unscrew the jar and clean it with deionized water. Rinse again using the anode buffer. Then fill the 67 ml of anode buffer in the jar. Ensure the electrode is immersed inside the jar when screwing the jar back into the instrument.
Steps to Operate DNA Analyzer
Once the parts and instrument of the DNA analyzer are set up correctly and the required calibration is performed, the following steps are performed to run a successful sequencing cycle:
- Firstly assemble the plate correctly.
- Then insert the plate inside the instrument.
- Open the software on the computer and schedule a run for desired date and time.
- Run the sequencing cycle. The DNA sequencing time depends on the type of sequencing performed; for example, a long read DNA sequencing can run for 120 minutes, and standard read DNA sequencing can run for 60 minutes.
- Real-time, run data history and electropherogram can be viewed during or after the completion of the sequencing cycle.
Uses of Genetic Analyzer
The DNA analyzer is a modern and advanced helpful equipment for performing DNA analysis in a different laboratory. It can be suitable for Sanger sequencing, genotyping, STR and SNP profiling, gene mutation analysis, etc. The following are the area of application of the DNA analyzer:
- Forensic science: Molecular technique, especially gene sequencing, is widely used in forensic science. So, replacing most manual DNA sequencing work with a DNA analyzer will reduce the time required. It will also decrease the chance of cross-contamination. DNA sampler is also helpful in analyzing different fragments of DNA.
- Microorganism-related research laboratories: Gene analysis of bacteria and viruses is usually a choice while researching microorganisms. So, a DNA analyzer can be an ideal substitute for tedious manual work.
- Cancer-related laboratories: The DNA sampler helps detect genetic mutation and will be helpful in cancer-related laboratories for diagnosing the root cause of cancer.
Benefits of DNA Analyzer
- Large quantity of sample processing: The capillary array helps in processing 48-96 samples simultaneously, so it helps in processing a large amount of the samples.
- Less time-consuming: Since it can process multiple samples and require significantly less manual work, it will consume less time. Likewise, it is an automated process; a worker can finish other manual labor when the machine runs in the background.
- More efficient: DNA analyzers require only trained human resources to obtain results from at least 48 samples in very little time. So it increases the efficiency of laboratories.
- Less cross-contamination: Since sample handling is automatic, the chance of cross-contamination is significantly less or even nil.
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