Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

The overall goal of this experiment is to perform a rapid and accurate genotyping of toxic puffer fish by analyzing small PCR amplicons. The edible part of puffer fish that are used to prepare the puffer fish dish vary with its species and some of the species contain toxins throughout the body. This muscle the database and masu and fin and birth skin of puffer fish can help in the genotyping of puffer fish from the standpoint of full safety food for all and for investigation.

To perform DNA extraction using a kit, first place 30 to 50 milligrams of fish tissue in a 0.5 or 1.5 milliliter microcentrifuge tube. Squash the tissue without fin and skin, using a microspatula. Add 180 microliters of the animal tissue lysis buffer and 40 microliters of proteinase k and vortex briefly.

Incubate at 56 degrees Celsius on a block heater for two hours or overnight. Centrifuge the sample for five minutes at 13000xg. After transferring the supernatant to a new 1.5 milliliter microcentrifuge tube add 200 microliters of the chaotropic buffer containing guanidine hydrochloride from the kit.

Then add 200 microliters of 99.5%ethanol and mix by vortexing. Transfer the mixture, including any precipitate, into the spin column placed in a two milliliter collection tube. After centrifuging for one minute at 13000xg, discard the flow-through.

Add 500 microliters of wash buffer one, containing guanidine hydrochloride from the kit, and centrifuge as before. Discard the flow-through and the collection tube. Place the spin column in a new two milliliter collection tube provided with the kit.

Add 500 microliters of wash buffer two and centrifuge for three minutes at 20, 000xg. After discarding the flow-through and the collection tube transfer the spin column to a new 1.5 milliliter centrifuge tube. Pipette 200 microliters of the elution buffer to the center of the spin column membrane.

After one minute, centrifuge for one minute at 6000xg. Then pipette 50 microliters of the aluette to a disposable ultraviolet cuvette. Measure the ultraviolet spectrum of the aluette at 220 to 300 nanometers and the absorbance at 260 nanometers using a spectrophotometer.

Using detergent free reagent is critical for the success of liquid chromatography because reagent cause indeed possible damage to the analytical column. Working on a clean bench to avoid contamination, set up a 25 microliter PCR for each extracted DNA sample, a positive control and a negative control, on ice as described in the text protocol. For easy operation, make a sufficient amount of cocktail containing all reagents without template DNA and dispense it.

Use DNA polymerase having proofreading activity to avoid the addition of three prime adenine overhangs to the PCR product. Carryout PCR amplification on a thermal cycler using the cycle program shown in the text protocol. To perform the enzymatic digestion add two microliters of the enzymatic digestion buffer to the PCR solution.

Add one microliter of each restriction enzyme and mix gently. Incubate the sample for 30 minutes at 37 degrees Celsius in the thermal cycler, followed by five minutes at 72 degrees Celsius. Filter the reaction solution with a 0.2 to 0.5 micron centrifugal filter device to prevent clogging and avoid damaging the analytical column.

Transfer the filtrate to a tapered polypropylene vial and store at 20 degrees Celsius until analysis. Prepare a solution of hexafluoroisopropanol and triethylamine in a one liter bottle as described in the text protocol. Connect the bottle to line A of the liquid chromatography instrument, cooling the bottle using a direct cooling portable refrigerator to prevent vaporization.

Connect another bottle filled with methanol to line B.Connect the guard column and the analytical column to the instrument. Start flowing the initial mobile phase to equilibrate the columns. If you are using a mass spectrometer without software for controlling the C-Trap pressure you are required to reduce the pressure of the C-Trap By manually controlling the bar.

To do this, pull the knob to unlock, and turn the knob anti-clockwise very slowly to reduce the high vacuum pressure to 1/3. The indicated pressure should change gradually in a delayed manner. Ignore the warning message about low pressure.

Push the knob to lock. After setting up for the calibration, as described in the text protocol, move the heated electrospray ionization probe closer to the interface of the mass spectrometer at position B.Connect to the probe and a syringe with a tube. Infuse the calibrant constantly at a flow rate of 10 microliters per minute using the embedded syringe pump.

Check only the six lower mass ions in the customized calibration table. Proceed with calibration after the intensity of the signals are stabilized. After the calibration, check only the six higher mass ions and proceed with calibration.

Avoid calibrating using all ions at once to avoid failure. Move the probe to position D for a flow rate of 0.4 milliliters per minute and connect to the instrument with an inert metal tube. After setting up the parameters as described in the text protocol, purge bubbles by tapping the bottoms of the vials.

Set the sample vials on the sample rack and inject one microliter of a sample using the autosampler to start analysis. Open the raw data files with a browser software and confirm that all the acquisitions are successfully finished, including the positive and negative controls. Set up a method as described in the text protocol and then click the run button in the run cue tab to start deconvolution software.

After the cue is completed select a row and click open result in the upper caption to obtain the results of the deconvolution. Here is a typical total ion current chromatogram obtained from a negative control and a puffer fish sample. Each peak represents a DNA duplex which is separated based on their length.

In this example, three peaks from the puffer fish sample are observed. These peaks are the results of the amplicon digestion using two endonucleases. No peaks are observed in the negative control sample.

Each peak represents a mass spectrum that comprises multiply charged ions. Enlarging the mass spectrum enables dozens of isotopically resolved peaks to be noticeable. This resolution is a requisite for determining monoisotopic mass and is only feasible via a high resolution mass spectrometer.

In this example, the monoisotopic masses of the third peak are 16296.7656 and 16468.6616 Daltons. Considering a mass tolerance of three ppm, the measured values are identical to the theoretical values of takifugu rubripes as shown here. Once mastered, this technique can be done in one day using common instruments and apparatus.

After watching this video you should have a good understanding of how to analyze small amplicons by liquid chromatography high resolution mass spectrometry for genotyping. Technique can be applied in analyzing other DNA polymorphisms provided the sequence variation is reflected in the small amplicons to be analyzed. However, this technique identifies only base compositions, base substitution within the same mercure can't be distinguished.