Pyrosequencing: A Simple Method for Accurate Genotyping

Genetics research has benefited greatly from the release of the human genome sequence. Pyro sequencing is a unique system that allows the analysis of genetic variation as well as RNA Allelic imbalance, DNA, methylation status and gene copy number. In this video, we demonstrate a basic pyro sequencing reaction for genetic analysis.

Hi, I'm Kristy King from the laboratory of Dr.Charles Eby and Dr.Brian Gage in the Department of Internal Medicine at Washington University School of Medicine. Today we are gonna show you a procedure for pyro sequencing. This procedure is useful because it's one of the most thorough yet simple methods used to analyze SNPs.

Pyro sequencing is based on sequencing by synthesis, taking advantage of the release of pyrophosphate whenever a nucleotide is incorporated in an open three prime DNA strand. Once a plate is loaded, nucleotides are incorporated based upon a sequence provided by the software Once released, the pyrophosphate is used in a reaction that results in the release of a TP, which is used by luciferase to convert Lucifer to oxy Lucifer resulting in the emission of light. The admitted light is collected by a CCD camera and recorded as peaks, also known as pyros.

Any nucleotides not incorporated into the DNA strand are degraded by AP pyra to prevent background noise. This procedure involves the following steps. Assay design, PCR gel electrophoresis for quality control and pyro sequencing.

So let's get started with pyro sequencing. In order To perform pyro sequencing, A PCR product must first be prepared. PCR for pyro sequencing requires more cycles than most PCRs in order to ensure that all of the primer is used up roughly 50 cycles.

Pyro sequencing also requires that one of the PCR primers is biotinylated. Lastly, it is important to note that an internal primer is also required. Efficient assay design may be carried out with software provided through pyro sequencing to ensure contamination is not present.

And to confirm PCR product is present, a gel should be run on a few samples as well as a negative control. To make the pyro sequencing plate add 12 microliters of the pyro sequencing primer mix to the 96 well pyro sequencing plate. This requires a mixture of 43.2 microliters of the internal primer, along with 1396.8 microliters of a kneeling buffer cover the pyro sequencing plate with adhesive film.

If the setup is to take longer than 50 minutes to each well of the 96 well PCR product add 70 microliters of SRO speed mix that contains 240 microliters streptavidin coated sero speeds. 4, 560 microliters of binding buffer, which is made of triss sodium chloride, EDTA and tween 20 and 3, 600 microliters of water and replace the lid securely. Place the 96 well PCR product plate with the bead mix in a plate shaker for five minutes at room temperature.

Make sure the lid is secured to prevent any cross-contamination between the wells. This step will allow thorough a kneeling of the strep ENC, coated spheros beads to the biotin tag that is located on the PCR primer. Set up a workstation for prepping the plates.

That includes the reagent troughs, PCR product and bead mix tray and the pyro sequencing primer tray. Make sure that the PCR product and bead mix plate as well as the pyro sequencing primer tray are properly aligned so that the negatives are in the same orientation. Before transferring samples, shake the vacuum tool, which is turned off in clean water to release any beads or debris that may be on it.

Discard the remaining water, refill the trough, and turn the vacuum on. Leave the vacuum tool in the trough until all water has been removed, which is approximately 30 seconds. Place the filter tips of the vacuum tool into the wells of the PCR bead mix plate and let it remain until all liquid has been removed from the plate.

Gentle rocking may be used to prevent surface tension. Place the vacuum in the 70%ethanol trough when the liquid begins to flow through the tubing. Allow the filter tips to suck up the ethanol for five seconds.

Repeat for the 0.2 molar sodium hydroxide, which denatures the DNA to single stranded PCR and for the washing buffer. To cleanse and neutralize the PCR product. Disconnect the vacuum hose from the vacuum tool and place the vacuum tool into the pyro sequencing plate containing the pyro sequencing primer in a kneeling buffer mix.

If the vacuum hose is still connected, when placed into the pyro sequencing plate, the primer mix will be sucked through. The tips causing you to lose your PCR product. Gently shake or rock the tips of the vacuum in the wells of the pyro sequencing plate to disperse your PCR product.

Remove the vacuum tool from the pyro sequencing plate once the shaking or rocking is complete, and reconnect the vacuum tool to the hose and place the tool into the clean water to cleanse it for the next plate. Place the pyro sequencing plate on a heat block for two minutes at 80 degrees Celsius. After two minutes, remove the plate from the heat block and place on a cool surface.

Once cooled and adhesive film can be used to cover the plate, unless the plate will be run within 15 minutes to prevent evaporation. And now it's time to begin pyro Sequencing. The first step of pyro sequencing is to enter the assay details into the computer.

Under simplex entry, select the new entry and enter assay information, including an ID name and the sequence to analyze, which is provided by the assay design software and allows for quality control select dispensation order, which provides the sequence around the SNP plus control bases. Lastly, select histograms to provide a visual of what your pigram should look like. Now it's time to enter a snip run.

Under the snip runs and the general tab, enter a run name and select instrument parameters for the run. Under the setup tab, select the assay entry and click and drag over the plate. To enter the assay of choice into your plate, it's important to note that the entire plate does not have to be used in which you can inactivate different wells for analysis, as well as many different assays may be run on the same plate.

Click the view tab and then select.Run. This page lists the appropriate volumes of nucleotides, enzyme, and substrate needed for the run. Clean both the nucleotide or capillary and reagent tips before use.

Fill the tips with water and apply pressure over the top of the tip to check for any tip blockages. If water does not squirt from the bottom of the tip, empty and refill several times to try to force the water through. Or you can sonicate the tips if the tip remains blocked.

Discard and get new tips. The enzyme and substrate should be resuspended with water before use. If shaken air bubbles could result causing tip blockages or inconsistent dispensation, unused resuspended enzyme and substrate can be stored at minus 20 degrees Celsius.

For future use for capillary dispensing tips in a micro fuge tube, make a one-to-one dilution with the nucleotides and te buffer P eight. Mix well before use. Fill the nucleotide and reagent tips with the appropriate volumes according to the amounts suggested by the software.

Gently dispense liquid down the sides of the tips to prevent pipetting air bubbles into them and causing blockages. Be sure to check for any air bubbles in the nucleotide dispensing tips. If any air bubbles are present, simply tap the sides of the tips until the air bubbles surface or dislodge them with a clean pipette tip.

Run a test plate after filling the cartridge. Place the cartridge in the pyro sequencing instrument and the test plate in the 96 well plate platform. Placing an adhesive film over the plate allows for the reagent and nucleotide dispensation to be easily seen.

Select the instrument tab on the left side of the screen, then click manage. Select the instrument from the dropdown menu. Click test, and a warning will appear.

Asking you to check the test plate has been placed in the instrument. Click okay. Once complete, remove the test plate and in the middle of the plate should be liquid dots over six of the wells representing four nucleotides, enzyme and substrate.

If there are less than six small dots, a blockage has occurred and the tips should be checked and removed of any blockages. Place the plate in the pyro sequencing 96 well plate platform. Close all levers and click run on the individual plate.

Run set up the enzyme substrate and nucleotides will dispense in the predetermined order. Once the run has been analyzed by the pyro sequencer, it's time to examine the run. Blue wells represent a passing genotype and pyro.

Orange wells call for human intervention and may be edited by clicking on the well of interest and opening the predicted histogram. A genotype may be passed, failed or checked, as well as the genotype itself changed appropriately. Once a well is edited, a dark circle will show on the plate map.

Negative controls should be scored as negative. There may be non-specific peaks in the negative wells, however, it's typically caused by looping of the internal primer. So we've Just shown you how to py sequence a polymorphism in human genomic.

DNA Pyro sequencing is useful over other sequencing procedures because it's adaptable to a wide range of applications. It's also robust enough to handle DNA from any source, including low concentration and degraded DNA. When doing this procedure, it's important to remember to start with a good clean PCR product.

Include a negative control for each assay and ensure all of your reagents are good. So that's it. Thanks for watching and good luck with your experiments.