The overall goal of the following experiment is to identify a bacterium through 16 s ribosomal DNA sequence analysis using the pyro sequencing methodology. This is achieved by first amplifying the target DNA through a polymerase chain reaction using a biotinylated primer. The biotinylated amplicon is then immobilized using streptavidin coated spheros beads and purified using wash buffers.
Next, the amplicon is denatured and 16 s ribosomal sequencing primers are ELL to the single strands of DNA. The goal of this step is to prepare the DNA for sequencing. Finally, the DNA sequencing reaction takes place by pyro sequencing, a rapid and accurate method to sequence nucleic acids based on the principle of sequencing by synthesis.
The biotinylated alicon is used as a template by DNA polymerase to incorporate DN TPS one base at a time leading to the generation of pyrophosphate. A TP Sul Urease proportionately converts pyrophosphate to A-T-P-A-T-P catalyzes the luciferase mediated conversion of Lucifer to Lucifer, which generates light that is proportional to the amount of a TP.The light is recorded as a peak on the pyro trace and indicates nucleotide incorporation. The unincorporated DN NTPs are degraded by apras before the next DNTP is added for continuation of the synthesis.
The chemiluminescent signal recorded on the rogram allows the sequence of DNA to be determined. The results obtained are ribosomal 16 SDNA sequences, which are used to identify the bacterium by comparison to a ribosomal DNA reference database. Pyro sequencing is a versatile technique that facilitates microbial genome sequencing and can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to antimicrobial agents.
In this video, the procedure for bacterial amplicon generation amplicon pyro sequencing and DNA sequence analysis will be demonstrated To begin the procedure for template amplification, thaw all required solutions and mix each one thoroughly. All work can be done at room temperature. Set up the PCR reaction according to this table.
Note that a final magnesium chloride concentration of 1.5 millimolar will provide satisfactory results in most cases. However, if a higher magnesium concentration is required up to 3.5 microliters of 25 millimolar, magnesium chloride can be added to a reaction. One primer must be biotinylated at its five prime end, and it's recommended that the primer be HPLC purified thoroughly.
Mix the reaction mixture by gently pipetting up and down, and then dispense appropriate volumes into PCR tubes. Next, add template DNA to each PCR tube. 10 nanograms of genomic DNA per reaction is recommended.
Place the PCR tubes in a cycler and start this cycling program prior to making the master mix. Swirl the bottle of strep avid and coated seros beads to ensure a homogenous suspension. Make a master mix according to this flow chart.
Combining beads, binding buffer, and high purity water in a micro centrifuge tube. Depending on the volume of PCR product used, dispensed 60 to 75 microliters of master mix into each well of a PCR plate so that the total volume is 80 microliters per well. Add five to 20 microliters of Biotinylated PCR product to each well.
The volume per well should now be 80 microliters. Seal the wells with strip caps and agitate the PCR plate at 1, 400 RPM for five to 10 minutes at room temperature using an orbital shaker. Begin this procedure by diluting the sequencing primers to 0.3 micromolar with a kneeling buffer and dispensing 25 microliters into each well of the reaction plate.
Position the plate on the vacuum workstation. Fill the workstation troughs according to this diagram. 50 milliliters of 70%ethanol in trough one 40 milliliters of denaturation solution in trough two 50 milliliters of wash buffer in trough three 50 milliliters of high purity water in trough four and 70 milliliters of high purity water in trough five.
Turn on the vacuum pump and make sure the switch on the vacuum tool is set to on flush the filter probes with high purity water. In trough five, position the PCR plate with beads on the workstation ensuring that both PCR and reaction plates are in the same orientation. Lower the vacuum tool into the wells of the PCR plate.
For 20 seconds or until all volume has been aspirated, the beads will be captured by the vacuum tool with the vacuum still on. Flush the tool with 70%ethanol for 20 seconds or until all volume has been aspirated. Next, flush the tool with Denaturation solution for 20 seconds or until all volume has been aspirated.
After that, flush the tool with wash buffer for 20 seconds or until all volume has been aspirated with the vacuum still on. Raise the vacuum tool to beyond 90 degrees vertical for five seconds to allow all liquid to drain out of the vacuum tool. Now turn the pump off and align the vacuum tool with three reaction plate.
Lower the vacuum tool into the wells. Then gently shake from side to side for approximately 10 seconds to release the beads into the wells containing sequencing primer. Place the vacuum tool in the water trough for cleaning to aneel the sequencing primer to the DNA.
Place the reaction plate in a prewarm plate holder. Heat the plate at 80 degrees Celsius for two minutes. Remove the plate from the holder and place it on the counter to cool it room temperature for five minutes.
Begin this procedure by switching on the instrument using the power switch located above the power cord to determine volumes needed. Set up a run file in the instrument software and under the tools menu, select pre-run information. Fill a cartridge according to the instructions provided in the pre-run information page.
Put the pipette tip in the corner of each well as far down as possible, ensuring that no air bubbles are present. Open the cartridge gate and insert the cartridge with the label facing forward. Ensure that the cartridge is properly inserted and close the gate.
Open the plate holding frame and position the reaction plate inside the instrument. Close the plate holding frame and the instrument lid. Insert the USB stick with the run files created with the instrument software into the USB port at the front of the instrument press.
Okay to see the menu of desired run files. Use the scroll arrows to select the desired run file and press select. When all preset pressure and temperature levels are reached, the instrument will begin dispensing reagents during a run.
The instrument screen displays the pyro of the selected well in real time. Use the scroll arrows to view the pyros of other wells. When the instrument confirms that the run is finished and the run file has been saved to the USB stick, press close.
Remove the USB stick. The results of a typical pyro sequencing run are shown here. Forward and reverse PCR primers are designed for conserved regions of the DNA template, which is the 16 s ribosomal sequence.
In this example, the sequencing primer is positioned immediately upstream of a well characterized identifying hypervariable DNA sequence within the amplicon shown in blue. This hypervariable sequence allows for bacterial identification of a large number of bacterial species using the conserved sequencing primer as an internal quality control, the DNA sequence bracketing the variable region can be checked to assure that the correct DNA region has been analyzed. The pyro sequencing software allows for comparison and alignment of the generated sequence to an internal database of bacterial ribosomal sequences for bacterial identification.
In addition, a sequence can be analyzed to determine mutations that confer antibiotic drug resistance. For example, analysis of mutations in the 23 s ribosomal genes of helicobacter pylori reveals two mutation patterns, GAA or a GA that confer antibiotic resistance. Pyro sequencing is a versatile technique that facilitates microbial genome sequencing.
The advantages of pyro sequencing from microbiology applications include rapid and reliable high throughput screening and accurate identification of microbes and microbial genome mutations. In addition, the pyro sequencing methodology can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs point mutations, insertions and deletions, as well as quantification of multiple gene copies that may occur in some antimicrobial resistance patterns. Thank you for watching this video and best of luck with your sequencing experiments.
